Grain size is an important determinant of grain yield in rice. Although dozens of grain size genes have been reported, the molecular mechanisms that control grain size remain to be fully clarified. Here, we report the cloning and characterization of GR5 (GRAIN ROUND 5), which is allelic to SMOS1/SHB/RLA1/NGR5 and encodes an AP2 transcription factor. GR5 acts as a transcriptional activator and determines grain size by influencing cell proliferation and expansion. We demonstrated that GR5 physically interacts with five Gγ subunit proteins (RGG1, RGG2, DEP1, GS3, and GGC2) and acts downstream of the G protein complex. Four downstream target genes of GR5 in grain development (DEP2, DEP3, DRW1, and CyCD5;2) were revealed and their core T/CGCAC motif identified by yeast one-hybrid, EMSA, and ChIP-PCR experiments. Our results revealed that GR5 interacts with Gγ subunits and cooperatively determines grain size by regulating the expression of downstream target genes. These findings provide new insight into the genetic regulatory network of the G protein signaling pathway in the control of grain size and provide a potential target for high-yield rice breeding.

BACKGROUND: Babesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.
METHODS: In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.
RESULTS: Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.
CONCLUSIONS: This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.

BACKGROUND: WRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown.
RESULTS: In this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds.
CONCLUSIONS: Together, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs.

Heading date is crucial for rice reproduction and the geographical expansion of cultivation. We fine-mapped qHD5 and identified LOC_Os05g03040, a gene that encodes an AP2 transcription factor, as the candidate gene of qHD5 in our previous study. In this article, using two near-isogenic lines NIL(BG1) and NIL(XLJ), which were derived from the progeny of the cross between BigGrain1 (BG1) and Xiaolijing (XLJ), we verified that LOC_Os05g03040 represses heading date in rice through genetic complementation and CRISPR/Cas9 gene-editing experiments. Complementary results showed that qHD5 is a semi-dominant gene and that the qHD5XLJ and qHD5BG1 alleles are both functional. The homozygous mutant line generated from knocking out qHD5XLJ in NIL(XLJ) headed earlier than NIL(XLJ) under both short-day and long-day conditions. In addition, the homozygous mutant line of qHD5BG1 in NIL(BG1) also headed slightly earlier than NIL(BG1). All of these results show that qHD5 represses the heading date in rice. Transient expression showed that the qHD5 protein localizes to the nucleus. Transactivation activity assays showed that the C-terminus is the critical site that affects self-activation in qHD5XLJ. qRT-PCR analysis revealed that qHD5 represses flowering by down-regulating Ehd2. qHD5 may have been selected during indica rice domestication.

The combination of apomixis and hybrid production is hailed as the holy grail of agriculture for the ability of apomixis to fix heterosis of F1 hybrids in succeeding generations, thereby eliminating the need for repeated crosses to produce F1 hybrids. Apomixis, asexual reproduction through seed, achieves this feat by circumventing two processes that are fundamental to sexual reproduction (meiosis and fertilization) and replacing them with apomeiosis and parthenogenesis, resulting in seeds that are clonal to the maternal parent. Parthenogenesis, embryo development without fertilization, has been genetically engineered in rice, maize, and pearl millet using PsASGR-BABY BOOM-like (PsASGR-BBML) transgenes and in rice using the OsBABY BOOM1 (OsBBM1) cDNA sequence when expressed under the control of egg cell-specific promoters. A phylogenetic analysis revealed that BABY BOOM (BBM)/BBML genes from monocots cluster within three different clades. The BBM/BBML genes shown to induce parthenogenesis cluster within clade 1 (the ASGR-BBML clade) along with orthologs from other monocot species, such as Setaria italica. For this study, we tested the parthenogenetic potential of three BBM transgenes from S. italica, each a member of a different phylogenetic BBM clade. All transgenes were genomic constructs under the control of the AtDD45 egg cell-specific promoter. All SiBBM transgenes induced various levels of parthenogenetic embryo development, resulting in viable haploid T1 seedlings. Poor seed set and lower haploid seed production were characteristics of multiple transgenic lines. The results presented in this study illustrate that further functional characterization of BBMs in zygote/embryo development is warranted.

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

Fragaria × ananassa Duch, which among the youngest fruit crops, comprises many popular cultivars that are famous for their favored color and aroma. The regulation roles of AP2/ERF (APETALA2/ethylene-responsive element-binding factor) transcription factors in fruit flavor and color regulation have been studied in several fruit crops. The AP2 family of strawberry, which was ignored in recent AP2/ERF identification studies, was explored in this study. A total of 64 FaAP2 (Fragaria × ananassa AP2) transcription factors belonging to the euAP2, euANT (AINTEGUMENTA), and baselANT groups were identified with canonical insertion motifs in two AP2 domains. The motif identification illustrated that motifs 1, 5, and 2 indicated a corresponding AP2 domain repeat 1 with a linker region, and motifs 6, 4, 3 indicated a corresponding AP2 domain repeat 2, all of which were highly conserved. By synteny analysis, FaAP2 paralogs were identified in each sub-genome, and FaAP2 gene duplication and loss explained the unequal AP2 loci of sub-genomes. The expression profile in three cultivars indicated that six FaAP2 paralogs-four WRI (WRINKLED) gene homologs and two AP2 gene homologs-were candidate regulators of red fruit color and/or special fruit aroma. All these finds provide a basis for further investigations into role of AP2 in fruit color and aroma and would be helpful in the targeted selection of strawberry fruit quality to improve breeding.

Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.
Toxoplasma gondii is a single-celled parasite that can infect most warm-blooded animals, but only reproduces sexually in domestic and wild cats. Distantly related to the malaria agent, it currently infects over a quarter of the world’s human population. Although it is benign in most cases, the condition can still be dangerous for foetuses and people whose immune system is compromised. In the human body, Toxoplasma cells infiltrate muscle and nerve cells; there it undergoes a complex transformation that helps the parasites to stop dividing quickly and instead hide from the immune system in a dormant state. It is still unclear how this transition unfolds, and in particular which genes are switched on and off at any given time. To understand this transformation, scientists often measure which genes are active across a group of parasites. However, this approach gives only an ‘average’ picture and does not allow each parasite to be profiled, missing out on the diversity that may exist between individuals. One area of particular interest, for example, is a set of genes called SAG1-related sequences. They code for the ‘molecular overcoat’ of the parasite, an array of proteins that sit on the surface of Toxoplasma cells. More than 120 SAG1-related genes exist in the genome of each Toxoplasma parasite, creating a whole wardrobe of proteins that potentially hide the parasites from the immune system. Here, Xue et al. harnessed a technique called single-cell RNA sequencing, which allowed them to screen which genes were active in 5,400 individual Toxoplasma parasites from different strains. The analysis included both the rapidly dividing form of the parasite (present in the initial stage of an infection), and the slowly dividing form found in people who carry Toxoplasma without any symptoms. The resulting ‘atlas’ contains previously hidden information about the genes used at each stage of parasite development: this included unexpected similarities between Toxoplasma and the malaria agent, as well as subtle differences between two of the Toxoplasma strains. The atlas also sheds light on how individual parasites turns on SAG1-related sequences. It reveals a surprising diversity in the composition of the protein coats sported by Toxoplasma cells at the same developmental stage, a strategy that may help to thwart the immune system. One individual parasite in particular had an unusual combination of coat and other proteins found in both the fast and slow-dividing human forms. This parasite had been grown in human cells, yet a closer analysis revealed that it had activated several genes (including ones encoding the protein coat) that are normally only ‘on’ in the parasites going through sexual reproduction in domestic and wild cats. This new data atlas helps to understand how Toxoplasma are transmitted to and grow within humans, which could aid the development of treatments. Ultimately, a better knowledge of these parasites could also bring new information about the agent that causes malaria.

CONCLUSIONS: An AP2 family gene CBX1 is involved in mycorrhizal symbiosis and growth of Lotus japonicus. APETALA 2 (AP2) transcriptional regulator is highly conserved in plants. CBX1 from Lotus japonicus is a member of AP2 family. AMF (Arbuscular mycorrhizal fungi) inoculation experiment demonstrated that expression of CBX1 was significantly induced by AMF. Further promoter analysis showed that the - 764 to - 498 bp region of the CBX1 promoter containing CTTC motif is the AMF responsive region. Functional analysis of cbx1 mutant suggested CBX1 is critical for mycorrhizal symbiosis, especially for arbuscule formation. Moreover, under noncolonized condition, overexpression of CBX1 reduced the root length of L. japonicus but increased the size of root system and shoot length, whereas cbx1 mutant reduced the root size and shoot length, but not effect on root length. In addition, cbx1 altered activity of monolignol biosynthetic gene and increased lignin levels. Collectively, these data indicated that CBX1 is a positive regulator of symbiotic activity and plays roles in the growth of L. japonicus.

OBJECTIVE: Expression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer.
METHODS: Expression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR.
RESULTS: mRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3-9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue.
CONCLUSIONS: In ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3-9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers.