Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Brachypodium distachyon, a new monocotyledonous model plant, has received wide attention in biological research due to its small genome and numerous genetic resources. Codon usage bias is an important feature of genes and genomes, and it can be used in transgenic and evolutionary studies. In this study, the nucleotide compositions and patterns of codon usage bias were calculated using Codon W. Additionally, an ENC plot, Parity rule 2 and correspondence analyses were used to explore the major factors influencing codon usage bias patterns. The numbers of hydrogen bonds and skews were used to analyze the GC trend in the 5′-ends of the coding sequences. The results showed that minor differences in the codon usage bias patterns were revealed by the ENC plot, Parity rule 2 and correspondence analyses. The analyses of the CG-skew and the number of hydrogen bonds showed a declining trend in the number of cytosines at the 5′-ends of the CDSs (from the 5′-ends to the 3′-ends), indicating that GC may play a major role in codon usage bias. In addition, our results laid a foundation for the study of codon usage bias patterns in Brachypodium genus and suggested that the GC plays a major role in determining these patterns.

Ipomoea plants possess important commercial, medicinal, and ornamental value. Molecular and morphological studies have confirmed that most species of this genus exhibit similar phenotypes but complex phylogenetic relationships. To date, limited information is available on these evolutionary relationships. In this study, systematic analysis of diverse species from Ipomoea was used to elucidate the relationships in this genus. To this end, we employed the concept of codon usage bias (CUB) to analyze the codon usage bias of five Ipomoea species such as effective number of codons (ENC) and GC content at the third synonym codon position (GC3s). Three types of plots including ENC-GC3s, parity rule 2 (PR2) and neutrality plots were employed to discover the factors determining CUB, and the frequency of hydrogen bonds and nucleotide were calculated to dissect changes in GC content at the 50-end of the coding sequence. Our results showed little distinctness in CUB among the five species, with a reduction of hydrogen bonds content at the 50-end (with similar changes in cytosines). In addition, optimal codons of Ipomoea aquatica ended with G or C, different from those of the other four species, which ended in A or T. These results may be useful for exploring the evolutionary relationships among this group, and for understanding the reasons for the variation among Ipomoea species. (c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Chlorophyll content is a pivotal agronomic trait that directly determines photosynthetic efficiency and visual quality in tomato (Solanum lycopersicum). Although positive regulators of chlorophyll biosynthesis have been extensively characterized, the mechanisms of their transcriptional repression remain less understood. Here, we report that the GRAS family transcription factor SlGRAS17 functions as a key negative regulator of chlorophyll accumulation in tomato. We isolated a high-chlorophyll mutant, hcm1, from an ethyl methanesulfonate (EMS)-mutagenized population and identified the causative lesion as a premature stop codon in SlGRAS17. Both CRISPR/Cas9-generated slgras17 mutants and the original hcm1 allele exhibited dark green leaves with significantly elevated chlorophyll content, whereas SlGRAS17-overexpressing plants displayed a pale green phenotype with reduced chlorophyll levels. We demonstrated that SlGRAS17 localizes to the nucleus and directly binds to a distal region of the GOLDEN-like 1 (SlGLK1) promoter to repress its transcription. Genetic evidence from virus-induced gene silencing confirmed that the high-chlorophyll phenotype of slgras17 mutants is dependent on SlGLK1. Furthermore, we elucidated that SlGRAS17 physically interacts with the transcriptional co-repressor SEUSS (SlSEU3), which, in turn, bridges the interaction with LEUNIG (SlLUG) in a configuration that is proposed to form a complex with repressive function. Genetic analyses revealed that SlLUG is essential for SlGRAS17-mediated repression, as the loss of SlLUG abolished the repressive effect of SlGRAS17 overexpression on chlorophyll accumulation. Our study unveils a previously unidentified transcriptional regulatory module in which SlGRAS17 recruits the SlSEU3-SlLUG co-repressor complex to suppress SlGLK1 expression, thereby fine-tuning chlorophyll biosynthesis in tomato.