Identification of a highly efficient chloroplast-targeting peptide for plastid engineering.

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

Levels of photoactivated phototropin modulate signal transmission during the chloroplast accumulation response.

Chloroplasts accumulate in regions of plant cells exposed to irradiation to maximize light reception for efficient photosynthesis. This response is mediated by the blue-light receptor phototropin. Upon the perception of blue light, phototropin is photoactivated, an unknown signal is transmitted from the photoactivated phototropin to distant chloroplasts, and the chloroplasts begin their directional movement. How activated phototropin initiates this signal transmission is unknown. Here, using the liverwort Marchantia polymorpha, we analysed whether increased photoactive phototropin levels mediate signal transmission and chloroplast behaviour during the accumulation response. The signal transmission rate was higher in transgenic cells overexpressing phototropin than in wild-type cells. However, the chloroplast directional movement was similar between wild-type and transgenic cells. Consistent with the observation, increasing the amount of photoactivated phototropin through higher blue-light intensity also accelerated signal transmission but did not affect chloroplast behaviour in wild-type cells. Photoactivation of phototropin under weak blue-light led to the greater protein level of phosphorylated phototropin in cells overexpressing phototropin than in wild-type cells, whereas the autophosphorylation level within each phototropin molecule was similar. These results indicate that the abundance of photoactivated phototropin modulates the signal transmission rate to distant chloroplasts but does not affect chloroplast behaviour during the accumulation response.

JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI1 proteins in tomato.

In tomato, perception of jasmonates by a receptor complex, which includes the F-box protein CORONATINE INSENSITIVE 1 (COI1), elicits biosynthesis of defensive steroidal glycoalkaloids (SGAs) via a jasmonate-responsive ERF transcription factor, JRE4/GAME9. Although JRE4 is upregulated by jasmonate and induces the expression of many metabolic genes involved in SGA biosynthesis, it is not known whether JRE4 alone is sufficient for increased SGA biosynthesis upon activation of jasmonate signaling. Here, we show that application of methyl jasmonate induces the expression of JRE4 and SGA biosynthesis genes in leaves and hairy roots of wild-type tomato, but not in jasmonic acid insensitive 1 (jai1), a loss-of-function mutant allele of the tomato COI1 gene. Induced overexpression of JRE4 increased the expression of SGA biosynthesis genes in transgenic hairy roots of both wild-type tomato and the jai1 mutant, suggesting that JRE4 is the primary transcription factor that functions downstream of the jasmonate signaling pathway.

Jasmonate-Responsive ERF Transcription Factors Regulate Steroidal Glycoalkaloid Biosynthesis in Tomato.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

Light-dependent chloroplast relocation in wild strawberry (Fragaria vesca).

Chloroplast photorelocation is a vital organellar response that optimizes photosynthesis in plants amid fluctuating environmental conditions. Chloroplasts exhibit an accumulation response, in which they move toward weak light to enhance photoreception, and an avoidance response, in which they move away from strong light to avoid photodamage. Although chloroplast photorelocation has been extensively studied in model plants such as Arabidopsis thaliana, little is known about this process in the economically important crop strawberry. Here, we investigated chloroplast photorelocation in leaf mesophyll cells of wild strawberry (Fragaria vesca), a diploid relative of commercially cultivated octoploid strawberry (F. × ananassa). Microscopy observation revealed that the periclinal area of leaf mesophyll cells in F. vesca is considerably smaller than that of A. thaliana. Given this small cell size, we investigated chloroplast photorelocation in F. vesca by measuring light transmittance in leaves. Weak blue light induced the accumulation response, whereas strong blue light induced the avoidance response. Unexpectedly, strong red light also induced the accumulation response in F. vesca. These findings shed light on chloroplast photorelocation as an intracellular response, laying the foundation for enhancing photosynthesis and productivity in Fragaria.

C-Terminal Domain Controls Protein Quality and Secretion of Spider Silk in Tobacco Cells.

The remarkable mechanical strength and extensibility of spider dragline silk spidroins are attributed to the major ampullate silk proteins (MaSp). Although fragmented MaSp molecules have been extensively produced in various heterologous expression platforms for biotechnological applications, complete MaSp molecules are required to achieve instinctive spinning of spidroin fibers from aqueous solutions. Here, a plant cell-based expression platform for extracellular production of the entire MaSp2 protein is developed, which exhibits remarkable self-assembly properties to form spider silk nanofibrils. The engineered transgenic Bright-yellow 2 (BY-2) cell lines overexpressing recombinant secretory MaSp2 proteins yield 0.6-1.3  µg L-1 at 22 days post-inoculation, which is four times higher than those of cytosolic expressions. However, only 10-15% of these secretory MaSp2 proteins are discharged into the culture media. Surprisingly, expression of functional domain-truncated MaSp2 proteins lacking the C-terminal domain in transgenic BY-2 cells increases recombinant protein secretion incredibly, from 0.9 to 2.8 mg L-1 per day within 7 days. These findings demonstrate significant improvement in the extracellular production of recombinant biopolymers such as spider silk spidroins using plant cells. In addition, the results reveal the regulatory roles of the C-terminal domain of MaSp2 proteins in controlling their protein quality and secretion.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana.

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

Simultaneous introduction of multiple biomacromolecules into plant cells using a cell-penetrating peptide nanocarrier.

Plant cells contain groups of biomolecules that participate together in a particular biological process. Exogenous codelivery of multiple biomolecules is an essential step for elucidation of the biological significance of these molecules and enables various biotechnological applications in plants. However, the currently existing biomolecule delivery methods face difficulties in delivering multiple components into plant cells, mediating transgene expression, and maintaining the stability of the numerous components and lead to delays in biomolecular function. Cell-penetrating peptides (CPPs) have demonstrated remarkable abilities to introduce diverse biomolecules into various plant species. Here, we employed the engineered CPP KH9-BP100 as a carrier to deliver multiple biomolecules into plant cells and performed a bimolecular fluorescence complementation assay to assess the simultaneous introduction of multiple biomolecules. We demonstrate that multiple biomolecule/CPP cargos can be simultaneously internalized by a particular plant cell, albeit with different efficiencies. We present a cutting-edge technique for codelivery of multiple biomolecules into plant cells that can be used for elucidation of functional correlations and for metabolic engineering.

A marine photosynthetic microbial cell factory as a platform for spider silk production.

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

Targeted Gene Delivery into Various Plastids Mediated by Clustered Cell-Penetrating and Chloroplast-Targeting Peptides.

The plastid is an organelle that functions as a cell factory to supply food and oxygen to the plant cell and is therefore a potential target for genetic engineering to acquire plants with novel photosynthetic traits or the ability to produce valuable biomolecules. Conventional plastid genome engineering technologies are laborious for the preparation of plant material, require expensive experimental instruments, and are time consuming for obtaining a transplastomic plant line that produces significant levels of the biomolecule of interest. Herein, a transient plastid transformation technique is presented using a peptide-based gene carrier. By formulating peptide/plasmid DNA complexes that combine the functions of both a cell-penetrating peptide and a chloroplast-targeting peptide, DNA molecules are translocated across the plant cell membrane and delivered to the plastid efficiently via vesicle formation and intracellular vesicle trafficking. A simple infiltration method enables the introduction of a complex solution into intact plants, and plastid-localized transgene expression is expeditiously observed in various types of plastids in differentiated cell types of several plants. The gene delivery technology thus provides a useful tool to rapidly engineer plastids in crop species.

Identification of genes regulated by a jasmonate- and salt-inducible transcription factor JRE3 in tomato.

In Solanum lycoperisicum (tomato), a transcription factor of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, JASMONATE-RESPONSIVE ERF 3 (JRE3), is a closest homolog of JRE4, a master transcriptional regulator of steroidal glycoalkaloid (SGA) biosynthesis. In tomato genome, JRE3 resides in a gene cluster with JRE4 and related JRE1, JRE2, and JRE5, while JRE6 exists as a singleton on a different chromosome. All of the JREs are induced by jasmonates (JAs), whereas sodium chloride (NaCl) treatment drastically increases the expression of the JREs except for JRE4 and JRE6. In this study, to get insights into the regulatory function of the JA- and NaCl-inducible JRE3, a series of genes upregulated by ß-estradiol-induced overexpression of JRE3 are identified with microarray analysis in transgenic tomato hairy roots. No gene involved in the SGA pathway has been identified through the screening, confirming the functional distinction between JRE3 and JRE4. Among the JRE3-regulated genes, we characterize the stress-induced expression of genes encoding malate synthase and tonoplast dicarboxylate transporter both involved in malate accumulation. In transient transactivation assay, we reveal that both terminal regions of JRE4, but not a central DNA-binding domain, are indispensable for the induction of a gene involved in the JRE4 regulon. Functional differentiation of the JREs is discussed.

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.