Storage Proteins Of Maize Research Articles

Towards microalga-based superfoods: heterologous expression of zeolin in Chlamydomonas reinhardtii.

Microalgae are unicellular photosynthetic organisms that can be grown in artificial systems to capture CO2, release oxygen, use nitrogen- and phosphorus-rich wastes, and produce biomass and bioproducts of interest including edible biomass for space exploration. In the present study, we report a metabolic engineering strategy for the green alga Chlamydomonas reinhardtii to produce high-value proteins for nutritional purposes. Chlamydomonas reinhardtii is a species approved by the U.S. Food and Drug Administration (FDA) for human consumption, and its consumption has been reported to improve gastrointestinal health in both murine models and humans. By utilizing the biotechnological tools available for this green alga, we introduced a synthetic gene encoding a chimeric protein, zeolin, obtained by merging the γ-zein and phaseolin proteins, in the algal genome. Zein and phaseolin are major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris) that accumulate in the endoplasmic reticulum (ER) and storage vacuoles, respectively. Seed storage proteins have unbalanced amino acid content, and for this reason, need to be complemented with each other in the diet. The chimeric recombinant zeolin protein represents an amino acid storage strategy with a balanced amino acid profile. Zeolin protein was thus efficiently expressed in Chlamydomonas reinhardtii; thus, we obtained strains that accumulate this recombinant protein in the endoplasmic reticulum, reaching a concentration up to 5.5 fg cell-1, or secrete it in the growth medium, with a titer value up to 82 µg/L, enabling the production of microalga-based super-food.

Release kinetics of [lidocainium][ibuprofenate] as Active Pharmaceutical Ingredient-Ionic Liquid from a plasticized zein matrix in simulated digestion

An in vitro approach is proposed to study the release of an Active Pharmaceutical Ingredient-Ionic Liquid (API-IL) from a natural biopolymer matrix based on zein, a maize storage protein. Zein can be processed in the molten state with 20 w% [Lidocainium][Ibuprofenate] added as API-IL also acting as plasticizer and potentially co-plasticized by glycerol. The thermal stability of the matrix is checked, as well as the in vivo biological activity of the API-IL confirming anesthetic and anti-inflammatory activities. Model tablets are thermomolded at 130 °C (∅20 mm, 0.2 mm thick) and submitted to simulated digestion based on the INFOGEST static protocol of gastrointestinal food digestion at 37 °C (2 h under gastric conditions followed by 2 h under intestinal ones). The release of the API-IL is evaluated by HPLC-UV to dissociate lidocainium, that shows a progressive release (35 % after 2 h and 60 % after 4 h digestion), from ibuprofenate, that is mainly released under intestinal conditions due to low solubility in acidic conditions. The monitoring of the tablets reveals release mechanisms based on diffusion without noticeable erosion of the matrix. These results demonstrate the interest of this thermoplastic material to provide a relevant drug delivery system.

Microparticles and Nanoparticles from Plants—The Benefits of Bioencapsulation

The efficacy of drugs and vaccines depends on their stability and ability to interact with their targets in vivo. Many drugs benefit from encapsulation, which protects them from harsh conditions and allows targeted delivery and controlled release. Although many encapsulation methods are inexpensive, such as the formulation of tablets for oral delivery, others require complex procedures that add significantly to production costs and require low-temperature transport and storage, making them inaccessible in developing countries. In this review we consider the benefits of encapsulation technologies based on plants. Plant-derived biopolymers such as starch and the maize storage protein zein are already used as protective coatings, but plant cells used as production host provide natural in vivo bioencapsulation that survives passage through the stomach and releases drugs in the intestine, due to the presence of microbes that can digest the cell wall. Proteins can also be encapsulated in subcellular compartments such as protein bodies, which ensure stability and activity while often conferring additional immunomodulatory effects. Finally, we consider the incorporation of drugs and vaccines into plant-derived nanoparticles assembled from the components of viruses. These are extremely versatile, allowing the display of epitopes and targeting peptides as well as carrying cargoes of drugs and imaging molecules.

Viscoelastic properties of wheat gluten in a molecular dynamics study

Wheat (Triticum spp.) gluten consists mainly of intrinsincally disordered storage proteins (glutenins and gliadins) that can form megadalton-sized networks. These networks are responsible for the unique viscoelastic properties of wheat dough and affect the quality of bread. These properties have not yet been studied by molecular level simulations. Here, we use a newly developed α-C-based coarse-grained model to study ∼ 4000-residue systems. The corresponding time-dependent properties are studied through shear and axial deformations. We measure the response force to the deformation, the number of entanglements and cavities, the mobility of residues, the number of the inter-chain bonds, etc. Glutenins are shown to influence the mechanics of gluten much more than gliadins. Our simulations are consistent with the existing ideas about gluten elasticity and emphasize the role of entanglements and hydrogen bonding. We also demonstrate that the storage proteins in maize and rice lead to weaker elasticity which points to the unique properties of wheat gluten.

Artificial ER-Derived Vesicles as Synthetic Organelles for in Vivo Compartmentalization of Biochemical Pathways.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis,cis-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.

A SnRK1-ZmRFWD3-Opaque2 Signaling Axis Regulates Diurnal Nitrogen Accumulation in Maize Seeds.

Zeins are the predominant storage proteins in maize (Zea mays) seeds, while Opaque2 (O2) is a master transcription factor for zein-encoding genes. How the activity of O2 is regulated and responds to external signals is yet largely unknown. Here, we show that the E3 ubiquitin ligase ZmRFWD3 interacts with O2 and positively regulates its activity by enhancing its nuclear localization. Ubiquitination of O2 enhances its interaction with maize importin1, the α-subunit of Importin-1 in maize, thus enhancing its nuclear localization ability. We further show that ZmRFWD3 can be phosphorylated by a Suc-responsive protein kinase, ZmSnRK1, which leads to its degradation. We demonstrated that the activity of O2 responds to Suc levels through the ZmSnRK1-ZmRFWD3-O2 signaling axis. Intriguingly, we found that Suc levels, as well as ZmRFWD3 levels and the cytonuclear distribution of O2, exhibit diurnal patterns in developing endosperm, leading to the diurnal transcription of O2-regulated zein genes. Loss of function in ZmRFWD3 disrupts the diurnal patterns of O2 cytonuclear distribution and zein biosynthesis, and consequently changes the C/N ratio in mature seeds. We therefore identify a SnRK1-ZmRFWD3-O2 signaling axis that transduces source-to-sink signals and coordinates C and N assimilation in developing maize seeds.

3D Electron Microscopy Gives a Clue: Maize Zein Bodies Bud From Central Areas of ER Sheets.

Zeins are the main storage proteins in maize seed endosperm, and the onset of zein synthesis in young seeds challenges the endomembrane system and results in the formation of storage organelles. Even though zeins lack a conventional endoplasmic reticulum (ER) retention signal, they accumulate within the ER and assemble in conspicuous ER-derived protein bodies (PBs) stabilized by disulfide bridge formation and hydrophobic interaction between zein chains. Zein body formation during seed development has been extensively studied, as well as the mechanisms that lead to the initiation of PBs. However, the exact course of the PB formation process and the spatial relationship with the ER remain unclear. The development of serial block face scanning electron microscopy (SBF-SEM) techniques that allow three-dimensional imaging combined with the high resolution of electron microscopy provides new perspectives on the study of the plant endomembrane system. Here, we demonstrate that (i) the ER of maize seeds is mainly formed by massive sheets and (ii) PBs are not budding from tubules or the edge of sheets, but protrude from the entire surface of the ER sheet.

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells.

The encapsulation of biopharmaceuticals into micro‐ or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N‐terminal sequence of the maize storage protein, γ‐zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N‐terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration‐based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen‐presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.

Impact of co-expression of maize 11 and 18 kDa δ-zeins and 27 kDa γ-zein in transgenic soybeans on protein body structure and sulfur amino acid content

The methionine-rich seed storage proteins of maize have been expressed in transgenic plants as a means to improve the overall sulfur amino acid content of seed. Previous attempts to increase the sulfur amino acid content of soybean seeds by this approach has met with limited success. It has been shown co-expression of different class of zeins can result in their stable accumulation in transgenic plants. In this study, conventional crosses between transgenic plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein were made to obtain plants that simultaneously express both the δ-zein and γ-zein. Transmission electron microscopic observation of thin-sections of transgenic soybean seeds revealed that the zeins accumulated in ER-derived protein bodies (PBs) which were found sparsely scattered in cytoplasm. The size of these PBs varied from 0.2 to 0.6 μm in soybean plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein. In contrast, soybeans co-expressing the 18 kDa δ-zein and 27 kDa γ-zein the PBs was 3–4 times larger. Electron microscopic observation also revealed the sequestration of PBs inside the vacuoles where they could be subjected to degradation by vacuolar proteases. Amino acid analysis of transgenic soybean individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein revealed only a minimal increase in the overall methionine content compared to the wild-type. In contrast, plants co-expressing 18 kDa δ-zein and 27 kDa γ-zein showed a significant increase (27%) in the methionine content compared to the control seeds.

Uniparental and transgressive expression of α-zeins in maize endosperm of o2 hybrid lines.

The α-zein gene family encodes the most abundant storage proteins of maize (Zea mays) endosperm. Members of this family are expressed in a parent-of-origin manner. To characterize this phenomenon further, we investigated the expression of a subset of α-zein polypeptides in reciprocal crosses between o2 lines that were characterized by a simplified α-zein pattern. Maize lines that suppressed the expression of α-zeins when used as female parents were identified. The suppression was cross-specific, occurring only when specific genetic backgrounds were combined. Four α-zein sequences that were sensitive to uniparental expression were isolated. Molecular characterization of these α-zeins confirmed that their expression or suppression depended on the genetic proprieties of the endosperm tissue instead of their parental origin. DNA methylation analysis of both maternally and paternally expressed α-zeins revealed no clear correlation between this epigenetic marker and parent-of-origin allelic expression, suggesting that an additional factor(s) is involved in this process. Genetic analyses revealed that the ability of certain lines to suppress α-zein expression was unstable after one round of heterozygosity with non-suppressing lines. Interestingly, α-zeins also showed a transgressive expression pattern because unexpressed isoforms were reactivated in both F2 and backcross plants. Collectively, our results suggest that parent-of-origin expression of specific α-zein alleles depends on a complex interaction between genotypes in a manner that is reminiscent of paramutation-like phenomena.

The ZmbZIP22 Transcription Factor Regulates 27-kD γ-Zein Gene Transcription during Maize Endosperm Development.

Zeins are the most abundant storage proteins in maize (Zea mays) kernels, thereby affecting the nutritional quality and texture of this crop. 27-kD γ-zein is highly expressed and plays a crucial role in protein body formation. Several transcription factors (TFs) (O2, PBF1, OHP1, and OHP2) regulate the expression of the 27-kD γ-zein gene, but the complexity of its transcriptional regulation is not fully understood. Here, using probe affinity purification and mass spectrometry analysis, we identified ZmbZIP22, a TF that binds to the 27-kD γ-zein promoter. ZmbZIP22 is a bZIP-type TF that is specifically expressed in endosperm. ZmbZIP22 bound directly to the ACAGCTCA box in the 27-kD γ-zein promoter and activated its expression in wild tobacco (Nicotiana benthamiana) cells. 27-kD γ-zein gene expression was significantly reduced in CRISPR/Cas9-generated zmbzip22 mutants. ChIP-seq (chromatin immunoprecipitation coupled to high-throughput sequencing) confirmed that ZmbZIP22 binds to the 27-kD γ-zein promoter in vivo and identified additional direct targets of ZmbZIP22. ZmbZIP22 can interact with PBF1, OHP1, and OHP2, but not O2. Transactivation assays using various combinations of these TFs revealed multiple interaction modes for the transcriptional activity of the 27-kD γ-zein promoter. Therefore, ZmbZIP22 regulates 27-kD γ-zein gene expression together with other known TFs.

Engineering sulfur storage in maize seed proteins without apparent yield loss

Sulfur assimilation may limit the pool of methionine and cysteine available for incorporation into zeins, the major seed storage proteins in maize. This hypothesis was tested by producing transgenic maize with deregulated sulfate reduction capacity achieved through leaf-specific expression of the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase (EcPAPR) that resulted in higher methionine accumulation in seeds. The transgenic kernels have higher expression of the methionine-rich 10-kDa δ-zein and total protein sulfur without reduction of other zeins. This overall increase in the expression of the S-rich zeins describes a facet of regulation of these proteins under enhanced sulfur assimilation. Transgenic line PE5 accumulates 57.6% more kernel methionine than the high-methionine inbred line B101. In feeding trials with chicks, PE5 maize promotes significant weight gain compared with nontransgenic kernels. Therefore, increased source strength can improve the nutritional value of maize without apparent yield loss and may significantly reduce the cost of feed supplementation.

Divergent Transactivation of Maize Storage Protein Zein Genes by the Transcription Factors Opaque2 and OHPs.

Maize transcription factors (TFs) opaque2 (O2) and the O2 heterodimerizing proteins (OHP1 and OHP2) originated from an ancient segmental duplication. The 22-kDa (z1C) and 19-kDa (z1A, z1B, and z1D) α-zeins are the most abundant storage proteins in maize endosperm. O2 is known to regulate α-zein gene expression, but its target motifs in the 19-kDa α-zein gene promoters have not been identified. The mechanisms underlying the regulation of α-zein genes by these TFs are also not well understood. In this study, we found that the O2 binding motifs in the α-zein gene promoters are quite flexible, with ACGT being present in the z1C and z1A promoters and a variant, ACAT, being present in the z1B and z1D promoters. OHPs recognized and transactivated all of the α-zein promoters, although to much lower levels than did O2. In the presence of O2, the suppression of OHPs did not cause a significant reduction in the transcription of α-zein genes, but in the absence of O2, OHPs were critical for the expression of residual levels of α-zeins. These findings demonstrated that O2 is the primary TF and that OHPs function as minor TFs in this process. This relationship is the converse of that involved in 27-kDa γ-zein gene regulation, indicating that the specificities of O2 and the OHPs for regulating zein genes diverged after gene duplication. The prolamine-box binding factor by itself has limited transactivation activity, but it promotes the binding of O2 to O2 motifs, resulting in the synergistic transactivation of α-zein genes.

Quantification and In Vitro Bioaccessibility of Selenium from Osborne Fractions of Selenium‐Rich Cereal Grains

Cereal crops cultivated in the seleniferous belt of Punjab, India, were observed to hyperaccumulate a significantly high concentration of selenium (20–123 µg/g). Selenium concentration (µg/g) in storage proteins of wheat, maize, and rice, namely, albumin (401, 280, and 29, respectively), globulin (264, 192, and 242, respectively), glutelin (563, 359, and 178, respectively), and prolamin (629, 339, and 257, respectively) indicated variable selenium levels, with prolamin contributing significantly higher levels of selenium when compared with other proteins with reference to the total concentration of the protein fraction. The simulated gastric and gastrointestinal digestion studies indicated better accessibility of selenium during intestinal digestion, with variability across proteins and cereal types. The observations provide an insight into the bioavailability of selenium in selenium‐rich cereal grains, used in the study, and their potential use as source for selenium supplementation to deficient populations, or as “bioactive” selenium‐rich nutraceutical blends for health benefits.

Identification and Characterization of Maizefloury4as a Novel Semidominant Opaque Mutant That Disrupts Protein Body Assembly

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19-kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ER-associated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 α-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

Propriedades mecânicas e molhabilidade de filmes de zeínas extraídas de glúten de milho

Zeínas são proteínas de reserva do milho (prolaminas), que podem ser extraídas através de solubilização em meio alcoólico. Essas proteínas são altamente hidrofóbicas, com elevado grau de polimerização. Neste estudo, zeínas do tipo a (massa molecular entre 10 e 22 kDa) foram extraídas do glúten do milho e caracterizadas com respeito às suas principais bandas de absorção espectroscópica na região do infravermelho. Blendas de zeínas com ácido oléico (AO) como plastificante foram avaliadas em diversas proporções e filmes processados por casting sobre superfície apolar. Filmes de zeínas sem plastificante mostraram-se inviáveis para manipulação e medidas, mas a adição de 1 e 2% em massa de AO proporcionou um significativo aumento do módulo de elasticidade, segundo análise realizada por DMA. A presença de plastificante introduz também irregularidades superficiais e leva a uma perda das características hidrofóbicas dessas proteínas.

Handbook of maize: genetics and genomics

Handbook of maize: genetics and genomics

Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves.

BackgroundThe N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.ResultsWe analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.ConclusionsThis study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

BackgroundXylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).Methodology/Principal FindingsTobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.Conclusion/SignificanceIn the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.