The Intragenesis and Synthetic Biology Approach towards Accelerating Genetic Gains on Strawberry: Development of New Tools to Improve Fruit Quality and Resistance to Pathogens

Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis)

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

A CCaMK/Cyclops response element in the promoter of L. japonicus Calcium-Binding Protein 1 (CBP1) mediates transcriptional activation in root symbioses

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

Functional Analysis of the Lupinus luteus Cyclophilin Gene Promoter Region in Lotus japonicus

Functional analysis of promoter sequences is important to understand the regulation of gene expression. This study aimed to investigate the promoter region of the Lupinus luteus cytoplasmic cyclophilin gene (LlCyP; AF178458). After bioinformatic analysis, four promoter deletion fragments were fused to the β-glucuronidase reporter gene. We used Lotus japonicus as a model plant. After Agrobacterium rhizogenes transformation of L. japonicus, only the longest promoter region (−1055 bp to ATG) supported the β-glucuronidase expression in root nodule parenchyma. Putative cis-elements located between −1055 and −846 bp were subjected to site-directed mutagenesis. Mutations incorporated in the TGATT and AGATT motifs (cytokinin response) abolished GUS expression in nodules, but the mutated AAAGAT motif (OSE, organ-specific element) still activated the GUS expression in root nodules, mainly in cells surrounding the vascular bundle. Promoter deletion and mutation experiments suggest that cis-elements responsible for gene expression in the nodule are located in the region spanning from −1055 to −846 bp. We constructed a deletion fragment, in which the DNA sequence located between −822 and −198 bp was removed (pCYPMG). The promoter region arranged in the pCYPMG supports the expression in the parenchyma of L. japonicus nodules, but it is lower than the whole promoter region. The obtained results may be useful for transgene expression in determinate and indeterminate root nodules.

Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency

Phosphorus (P) deficiency is one of the major nutrient stresses restricting plant growth. The uptake of P by plants from soil is mainly mediated by the phosphate (Pi) transporters belonging to the PHT1 family. Multiple PHT1 genes from diverse plant species have been shown to be strongly up-regulated upon Pi starvation, however, the underlying mechanisms for the Pi-starvation-induced (PSI) up-regulation have not been well deciphered for most Pi transporter genes. Here, we reported a detailed dissection of the promoter activity of a PSI rice Pi transporter gene OsPT6, using the β-glucuronidase (GUS) reporter gene. OsPT6 promoter could drive GUS expression strongly in both roots and blades of rice plants grown under low P, but not high P. Cis-acting element analysis identified one copy of the P1BS motif and two copies of the W-box motif in OsPT6 promoter. Targeted deletion of the P1BS motif caused almost complete abolition of GUS induction in response to Pi starvation, irrespective of the presence or absence of the W-box motif, Four repeats of the P1BS motif fused to the CaMV35S minimal promoter was sufficient to induce GUS expression responsive to Pi starvation. Targeted deletion of the upstream W-box motif (W1) did not affect the GUS expression activity compared with the full-length OsPT6 promoter, while targeted deletion of the downstream W-box motif (W2) or both of the W-box motifs remarkably reduced the GUS induction rate upon Pi starvation. Our results proposed that the PSI response of OsPT6 was positively regulated by at least two elements, the sole P1BS and the downstream W-box, in its promoter, and the W-box-mediated up-regulation of OsPT6 might be highly dependent on the P1BS motif.

Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana

In the methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) is considered the key enzyme for the biosynthesis of terpenoids. In this study, PmDXS (MK970590) was isolated from Pinus massoniana. Bioinformatics analysis revealed homology of MK970590 with DXS proteins from other species. Relative expression analysis suggested that PmDXS expression was higher in roots than in other plant parts, and the treatment of P. massoniana seedlings with mechanical injury via 15% polyethylene glycol 6000, 10 mM H2O2, 50 μM ethephon (ETH), 10 mM methyl jasmonate (MeJA), and 1 mM salicylic acid (SA) resulted in an increased expression of PmDXS. pET28a-PmDXS was expressed in Escherichia coli TransB (DE3) cells, and stress analysis showed that the recombinant protein was involved in resistance to NaCl and drought stresses. The subcellular localization of PmDXS was in the chloroplast. We also cloned a full-length 1024 bp PmDXS promoter. GUS expression was observed in Nicotiana benthamiana roots, stems, and leaves. PmDXS overexpression significantly increased carotenoid, chlorophyll a, and chlorophyll b contents and DXS enzyme activity, suggesting that DXS is important in isoprenoid biosynthesis. This study provides a theoretical basis for molecular breeding for terpene synthesis regulation and resistance.

Promoter PPSP1–5-BnPSP-1 From Ramie (Boehmeria nivea L. Gaud.) Can Drive Phloem-Specific GUS Expression in Arabidopsis thaliana

Isolation of phloem-specific promoters is one of the basic conditions for improving the fiber development and resistance of ramie phloem using genetic engineering. In this study, we isolated a ramie endogenous promoter (named PPSP1-BnPSP-1) and analyzed the function of its truncated fragments in Arabidopsis. The results show that PPSP1-BnPSP-1 can drive the GUS reporter gene to be specifically expressed in the veins of Arabidopsis. After hormone and simulated drought treatment of the independent Arabidopsis lines carrying PPSP1-BnPSP-1 and its truncated fragments, only PPSP1–5-BnPSP-1 (−600 to −1 bp region of PPSP1-BnPSP-1) is stably expressed and exhibits phloem specificity. Our findings suggest that PPSP1–5-BnPSP-1 can be used as a phloem specific promoter for further research.

Genotypic Variability in Soybean [Glycine max (L.) Merrill] through Agrobacterium-Mediated Transformation

Embryonic tip explants of 92 Indian soyabean and 7 advanced breeding lines derived from soaked mature seeds were inoculated and co-cultivated for 5-day with Agrobacterium strain EHA105 carrying the binary vector pCambia1305.1 containing a hygromycin and kanamycin resistance gene as plant and bacterial selectable markers, respectively. Transient expression of transgene was monitored by histochemical localization of β-glucouronidase (GUSPlus) reporter activity in transformed ET tissues. A high genetic variability for Agrobacterium-infection ranging from 3.8 to 100% was observed in the form of transient GUS expression. Five highly efficient genotypes, namely DS-228, JS 335, JS 72-44, KHSb2, and JS 72-280 with transient GUS expression of 100, 98.1, 96.5, 96 and 92%, respectively were identified. In addition, various infectivity patterns in these genotypes were observed. Genotypes with very high transient GUS expression identified in this study may improve success rate of development of transgenic soybean. Plant Tissue Cult. & Biotech. 30(2): 231-242, 2020 (December)