Genomic Dissection and Diurnal Expression Analysis Reveal the Essential Roles of the PRR Gene Family in Geographical Adaptation of Soybean

Pseudo-response regulator (PRR) family members serve as key components of the core clock of the circadian clock, and play important roles in photoperiodic flowering, stress tolerance, growth, and the development of plants. In this study, 14 soybean PRR genes were identified, and classified into three groups according to phylogenetic analysis and structural characteristics. Real-time quantitative PCR analysis revealed that 13 GmPRRs exhibited obvious rhythmic expression under long-day (LD) and short-day (SD) conditions, and the expression of 12 GmPRRs was higher under LD in leaves. To evaluate the effects of natural variations in GmPRR alleles on soybean adaptation, we examined the sequences of GmPRRs among 207 varieties collected across China and the US, investigated the flowering phenotypes in six environments, and analyzed the geographical distributions of the major haplotypes. The results showed that a majority of non-synonymous mutations in the coding region were associated with flowering time, and we found that the nonsense mutations resulting in deletion of the CCT domain were related to early flowering. Haplotype analysis demonstrated that the haplotypes associated with early flowering were mostly distributed in Northeast China, while the haplotypes associated with late flowering were mostly cultivated in the lower latitudes of China. Our study of PRR family genes in soybean provides not only an important guide for characterizing the circadian clock-controlled flowering pathway but also a theoretical basis and opportunities to breed varieties with adaptation to specific regions and farming systems.     

Whole-Genome Sequencing and RNA-Seq Reveal Differences in Genetic Mechanism for Flowering Response between Weedy Rice and Cultivated Rice

Flowering is a key agronomic trait that influences adaptation and productivity. Previous studies have indicated the genetic complexity associated with the flowering response in a photoinsensitive weedy rice accession PSRR-1 despite the presence of a photosensitive allele of a key flowering gene Hd1. In this study, we used whole-genome and RNA sequencing data from both cultivated and weedy rice to add further insights. The de novo assembly of unaligned sequences predicted 225 genes, in which 45 were specific to PSRR-1, including two genes associated with flowering. Comparison of the variants in PSRR-1 with the 3K rice genome (RG) dataset identified unique variants within the heading date QTLs. Analyses of the RNA-Seq result under both short-day (SD) and long-day (LD) conditions revealed that many differentially expressed genes (DEGs) colocalized with the flowering QTLs, and some DEGs such as Hd1, OsMADS56, Hd3a, and RFT1 had unique variants in PSRR-1. Ehd1, Hd1, OsMADS15, and OsMADS56 showed different alternate splicing (AS) events between genotypes and day length conditions. OsMADS56 was expressed in PSRR-1 but not in Cypress under both LD and SD conditions. Based on variations in both sequence and expression, the unique flowering response in PSRR-1 may be due to the high-impact variants of flowering genes, and OsMADS56 is proposed as a key regulator for its day-neutral flowering response.     

Mechanisms of Vernalization-Induced Flowering in Legumes

Vernalization is the requirement for exposure to low temperatures to trigger flowering. The best knowledge about the mechanisms of vernalization response has been accumulated for Arabidopsis and cereals. In Arabidopsis thaliana, vernalization involves an epigenetic silencing of the MADS-box gene FLOWERING LOCUS C (FLC), which is a flowering repressor. FLC silencing releases the expression of the main flowering inductor FLOWERING LOCUS T (FT), resulting in a floral transition. Remarkably, no FLC homologues have been identified in the vernalization-responsive legumes, and the mechanisms of cold-mediated transition to flowering in these species remain elusive. Nevertheless, legume FT genes have been shown to retain the function of the main vernalization signal integrators. Unlike Arabidopsis, legumes have three subclades of FT genes, which demonstrate distinct patterns of regulation with respect to environmental cues and tissue specificity. This implies complex mechanisms of vernalization signal propagation in the flowering network, that remain largely elusive. Here, for the first time, we summarize the available information on the genetic basis of cold-induced flowering in legumes with a special focus on the role of FT genes.     

From Floral Induction to Blooming: The Molecular Mysteries of Flowering in Woody Plants

Flowering is a pivotal developmental process in response to the environment and determines the start of a new life cycle in plants. Woody plants usually possess a long juvenile nonflowering phase followed by an adult phase with repeated flowering cycles. The molecular mechanism underlying flowering regulation in woody plants is believed to be much more complex than that in annual herbs. In this review, we briefly describe the successive but distinct flowering processes in perennial trees, namely the vegetative phase change, the floral transition, floral organogenesis, and final blooming, and summarize in detail the most recent advances in understanding how woody plants regulate flowering through dynamic gene expression. Notably, the florigen gene FLOWERING LOCUS T(FT) and its antagonistic gene TERMINAL FLOWER 1 (TFL1) seem to play a central role in various flowering transition events. Flower development in different taxa requires interactions between floral homeotic genes together with AGL6 conferring floral organ identity. Finally, we illustrate the issues and corresponding measures of flowering regulation investigation. It is of great benefit to the future study of flowering in perennial trees.     

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.     

Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycine max). The Streptococcus pyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5′-NGG-3′ (SpCas9) or 5′-TTTN-3′ (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5′-NNGRRT-3′ (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5′-AAGGGT-3′, 5′-GGGGAT-3′, 5′-TTGAAT-3′, and 5′-TAGGGT-3′ in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.