Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.

Expression Changes of Genes Related to Germination Based on EST Database under Priming Treatment by Gibberellic Acid in Perilla frutescens (Korean Perilla)

It is very important to establish an optimal seed priming process in order to increase the vitality of the seeds and promote the metabolism for the germination of the seeds. The optimum concentrations and species of priming agents to improve seed germination of both medicinal plants were also estimated. To improve the germination rate of Perilla frutescens(Korean perilla) seeds, various seed priming agents were used to analyze seed germination rates in the Saeyeopsil, Okdong and 141 collection Korean perilla cultivars. The agents used for seed priming were CaCl2, Ca(NO3)2, NaCl, K3PO4, polyethylene glycol, and gibberellic acid (GA3). When 0.1 mMGA3was used for seed priming, germination rates of Okdong, and the 141 collection showed a greater than 70% increase compared to the controls. Nine genes were selected for expression analysis by searching for genes related to seed germination and plant development in the EST(Expressed Sequence Tag) database of the Korean perilla cDNA library. GA3priming treatment for 1 d induced higher transcriptional levels of genes related to germination and plant developmentthan controls treated with water only. These genes were identified as protochlorophyllide reductase-like, magnesium-chelatase subunit ChlI, heme-binding protein 2-like, glyceraldehyde 3-phosphate dehydrogenase A, Chlorophyll a-b binding protein 6, B2 protein, 2-Cys peroxiredoxin BAS1, and 21 kDa protein. From these results, we suggest that when priming Korean perilla seeds with GA3, a large number of genes involved in plant development at early stages of seed germination play a role in improving the seed germination rate. Also, these induced genes are ideal candidate biomarkers for seed priming of Korean perilla. Specially, protochlorophyllide reductase-like is thought to be a potential gene for future molecular marker.© 2021 Friends Science Publishers

Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

Establishment of A Rapid and Stable Infected System by Agrobacterium-Mediated Transformation of Germination Seeds in Diploid Strawberry.

Background Strawberry (Fragaria) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made in identification of gene function using stable and transient genetic transformation systems in strawberry, the limitation is, more or less, are present. To this end, development of a rapid, efficient, and stable transformation system is required for strawberry research and breeding. Results Here, using diploid Hawaii-4 (Fragaria vesca) seeds and a reporter gene of CHLH (the H subunit of magnesium chelatase magnesium chelatase) key to chlorophyll synthesis, we first develop a rapid, efficient, and stable infected system by the Agrobacterium-mediated seed infection to silence the reporter gene, reaching an infection frequency with 28.3% through a series of optimization elements, including seed full imbibition and initial germination, shaking infection for 24 h, dark cultivation on MS medium for 3 d at 24 ℃, light culture on MS-Tim medium for 1 week at 24 ℃, and vector construction tagged with fluorescence label. Taken together, radicle-emergence germination seeds, appropriate Agrobacterium concentration and infection time are critical for successful infection, finally obtaining the infected kanamycin-resistant seedlings of T1 generation by infected wild seeds within 1 month and T2 generation-infected plants within 4 months. Conclusions The Agrobacterium-mediated infection of germinating seeds (AMTGS) in diploid strawberry (F. vesca) is first established, providing a useful tool for gene function identification and improved agronomic traits in strawberry.

Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits

Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.

Establishment of a Rapid and Stable Transgenic System by Agrobacterium-Mediated Transformation of the Germination Seeds in Diploid Strawberry.

BackgroundStrawberry (Fragaria) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made in the identification of gene function using traditional, stable and transient genetic transformation systems in strawberry, the limitation is, more or less, present. Thus, development of a rapid, efficient, and stable transformation system is required for strawberry research and breeding.ResultsHere, using diploid Hawaii-4 (Fragaria vesca) seeds and a reporter gene of CHLH (the H subunit of magnesium chelatase magnesium chelatase) , we first develop a new, rapid, efficient, and stable transgenic system by the Agrobacterium-mediated seed transformation to silence the reporter gene, obtaining a transformation frequency with 10 % through a series of optimization conditions, including full imbibition and initial germination, shaking infection for 24 h, dark cultivation on MS medium for 3 d at 24 ℃, light culture on MS-Tim medium for 1 week at 24 ℃, and vector construction carried fluorescence label. Taken together, radicle-emergence germination seeds, appropriate Agrobacterium concentration and infection time are critical for successful transformation, obtaining transgenic kanamycin-resistant seedlings within 1 month and T2 generation transgenic plants within 4 months. ConclusionsWe first have successfully established Agrobacterium-mediated transformation of germinating seeds (AMTGS) in diploid strawberry (F. vesca), providing a useful tool for studying non-climacteric fruit ripening and strawberry breeding.

BEL1-LIKE HOMEODOMAIN4 regulates chlorophyll accumulation, chloroplast development, and cell wall metabolism in tomato fruit

Tomato (Solanum lycopersicum) is a model plant for studying fruit development and ripening. In this study, we found that down-regulation of a tomato bell-like homeodomain 4 (SlBL4) resulted in a slightly darker-green fruit phenotype and increased accumulation of starch, fructose, and glucose. Analysis of chlorophyll content and TEM observations was consistent with these phenotypes, indicating that SlBL4 was involved in chlorophyll accumulation and chloroplast formation. Ripened fruit of SlBL4-RNAi plants had noticeably decreased firmness, larger intercellular spaces, and thinner cell walls than the wild-type. RNA-seq identified differentially expressed genes involved in chlorophyll metabolism, chloroplast development, cell wall metabolism, and carotenoid metabolism. ChIP-seq identified (G/A) GCCCA (A/T/C) and (C/A/T) (C/A/T) AAAAA (G/A/T) (G/A) motifs. SlBL4 directly inhibited the expression of protoporphyrinogen oxidase (SlPPO), magnesium chelatase H subunit (SlCHLD), pectinesterase (SlPE), protochlorophyllide reductase (SlPOR), chlorophyll a/b binding protein 3B (SlCAB-3B), and homeobox protein knotted 2 (TKN2). In contrast, it positively regulated the expression of squamosa promoter binding protein-like colorless non-ripening (LeSPL-CNR). Our results indicate that SlBL4 is involved in chlorophyll accumulation, chloroplast development, cell wall metabolism, and the accumulation of carotenoids during tomato fruit ripening, and provide new insights for the transcriptional regulation mechanism of BELL-mediated fruit growth and ripening.