Genome-Wide Analysis of Cyclic Nucleotide-Gated Channel Genes Related to Pollen Development in Rice.

In the angiosperm, pollen germinates and rapidly expands the pollen tube toward the ovule. This process is important for plant double fertilization and seed setting. It is well known that the tip-focused calcium gradient is essential for pollen germination and pollen tube growth. However, little is known about the Ca2+ channels that play a role in rice pollen germination and tube growth. Here, we divided the 16 cyclic nucleotide-gated channel (CNGC) genes from rice into five subgroups and found two subgroups (clades II and III) have pollen-preferential genes. Then, we performed a meta-expression analysis of all OsCNGC genes in anatomical samples and identified three pollen-preferred OsCNGCs (OsCNGC4, OsCNGC5, and OsCNGC8). The subcellular localization of these OsCNGC proteins is matched with their roles as ion channels on the plasma membrane. Unlike other OsCNGCs, these genes have a unique cis-acting element in the promoter. OsCNGC4 can act by forming a homomeric complex or a heteromeric complex with OsCNGC5 or OsCNGC8. In addition, it was suggested that they can form a multi-complex with Mildew Resistance Locus O (MLO) protein or other types of ion transporters, and that their expression can be modulated by Ruptured Pollen tube (RUPO) encoding receptor-like kinase. These results shed light on understanding the regulatory mechanisms of pollen germination and pollen tube growth through calcium channels in rice.

Metabolomic Variability of Different Soybean Genotypes: ß-Carotene-Enhanced (Glycine max), Wild (Glycine soja), and Hybrid (Glycine max × Glycine soja) Soybeans.

We obtained a new hybrid soybean (Hybrid) by hybridizing ß-carotene-enhanced soybean (BCE; Glycine max L.) containing the phytoene synthase-2A-carotene desaturase gene and wild-type soybean (Wild; Glycine soja). To investigate metabolic changes between variants, we performed metabolic profiling of leaves (three growth stages) and seeds. Multivariate analyses revealed significant metabolic differences between genotypes in seeds and leaves, with seeds showing accumulation of phytosterols, tocopherols, and carotenoids (BCE only), indicating co-induction of the methylerythritol 4-phosphate and mevalonic acid pathways. Additionally, Hybrid produced intermediate levels of carotenoids and high levels of amino acids. Principal component analysis revealed metabolic discrimination between growth stages of soybean leaves and identified differences in leaf groups according to different genotypes at 8, 12, and 16 weeks, with Wild showing higher levels of environmental stress-related compounds relative to BCE and Hybrid leaves. The metabolic profiling approach could be a useful tool to identify metabolic links in various soybean cultivars.

Compositional equivalence assessment of insect-resistant genetically modified rice using multiple statistical analyses.

The safety of transgenic Bt rice containing bacteria-derived mCry1Ac gene from Bacillus thuringiensis (Bt) was assessed by conducting field trials at two locations for two consecutive years in South Korea, using the near-isogenic line comparator rice cultivar ('Ilmi', non-Bt rice) and four commercial cultivars as references. Compositional analyses included measurement of proximates, minerals, amino acids, fatty acids, vitamins, and antinutrients. Significant differences between Bt rice and non-Bt rice were detected; however, all differences were within the reference range. The statistical analyses, including analysis of % variability, analysis of similarities (ANOISM), similarity percentage (SIMPER) analysis, and permutational multivariate analysis of variance (PERMANOVA) were performed to study factors contributing to compositional variability. The multivariate analyses revealed that environmental factors more influenced rice components' variability than by genetic factors. This approach was shown to be a powerful method to provide meaningful evaluations between Bt rice and its comparators. In this study, Bt rice was proved to be compositionally equivalent to conventional rice varieties through multiple statistical methods.

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest ß-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.

Statistical study on the environmental effects on the natural variation of nutritional components in rice varieties.

This study was investigated to compare the natural variation of nutrients in rice variety by different environmental factors. Fifteen kinds of rices were used, which were cultivated in two locations for 2 years. All data were analyzed by the various statistical tools to identify the nutritional variations of nutrients. The results of variable importance in the prediction analysis were found to be consistent with the % variability. The nutrient compositions most affected by variety were fatty acids, and next were vitamins, proximate nutrients, minerals, and amino acids in order. The nutrient compositions most affected by location were proximate, followed by minerals, vitamins, fatty acids, and amino acids. For cultivation year, vitamins were most affected and then minerals, fatty acids, proximate nutrients, and amino acids in order. These findings could explain that each kind of nutrients can be naturally varied by different environmental factors.

The Pragmatic Introduction and Expression of Microbial Transgenes in Plants.

Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.

Exo-ethylene application mitigates waterlogging stress in soybean (Glycine max L.).

BACKGROUND: Waterlogging (WL) is a key factor hindering soybean crop productivity worldwide. Plants utilize various hormones to avoid various stress conditions, including WL stress; however, the physiological mechanisms are still not fully understood. RESULTS: To identify physiological mechanisms during WL stress, different phytohormones, such as ethephon (ETP; donor source of ethylene), abscisic acid, gibberellins, indole-3-acetic acid, kinetin, jasmonic acid, and salicylic acid were exogenously applied to soybean plants. Through this experiment, we confirmed the beneficial effects of ETP treatment. Thus, we selected ETP as a candidate hormone to mitigate WL. Further mechanistic investigation of the role of ETP in waterlogging tolerance was carried out. Results showed that ETP application mitigated WL stress, significantly improved the photosynthesis pigment, and increased the contents of endogenous GAs compared to those in untreated plants. The amino acid contents during WL stress were significantly activated by EPT treatments. The amino acid contents were significantly higher in the 100 µM ETP-treated soybean plants than in the control. ETP application induced adventitious root initiation, increased root surface area, and significantly increased the expressions of glutathione transferases and relative glutathione activity compared to those of non-ETP-treated plants. ETP-treated soybeans produced a higher up-regulation of protein content and glutathione S-transferase (GSTs) than did soybeans under the WL only treatment. CONCLUSIONS: In conclusion, the current results suggest that ETP application enabled various biochemical and transcriptional modulations. In particular, ETP application could stimulate the higher expression of GST3 and GST8. Thus, increased GST3 and GST8 induced 1) increased GSH activity, 2) decreased reactive oxygen species (ROS), 3) mitigation of cell damage in photosynthetic apparatus, and 4) improved phenotype consecutively.

A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana.

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

RASSF1A Directly Antagonizes RhoA Activity through the Assembly of a Smurf1-Mediated Destruction Complex to Suppress Tumorigenesis.

RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256-277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial-mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847-59. ©2016 AACR.

Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.

PPARδ promotes oncogenic redirection of TGF-ß1 signaling through the activation of the ABCA1-Cav1 pathway.

TGF-ß1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-ß1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-ß1 and plays a critical role in oncogenic redirection of TGF-ß1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-ß1-mediated growth inhibition, while its activation promotes TGF-ß1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-ß1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-ß1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-ß receptors (TßRs) and attenuates Smad but enhances ERK response to TGF-ß1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-ß1 and contributes to the oncogenic conversion of TGF-ß1 function through activation of the ABCA1-Cav1-TßR signaling axis.

Suppression of NS3 and MP is important for the stable inheritance of RNAi-mediated rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene.

Rice stripe virus (RSV) is a viral disease that seriously impacts rice production in East Asia, most notably in Korea, China, and Japan. Highly RSV-resistant transgenic japonica rice plants were generated using a dsRNAi construct designed to silence the entire sequence region of the RSV-CP gene. Transgenic rice plants were inoculated with a population of viruliferous insects, small brown planthoppers (SBPH), and their resistance was evaluated using ELISA and an infection rate assay. A correlation between the expression of the RSV-CP homologous small RNAs and the RSV resistance of the transgenic rice lines was discovered. These plants were also analyzed by comparing the expression pattern of invading viral genes, small RNA production and the stable transmission of the RSV resistance trait to the T3 generation. Furthermore, the agronomic trait was stably transmitted to the T4 generation of transgenic plants.

Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants.

Ethylene-responsive factors (ERFs), within a subgroup of the AP2/ERF transcription factor family, are involved in diverse plant reactions to biotic or abiotic stresses. Here, we report that overexpression of an ERF gene from Brassica rapa ssp. pekinensis (BrERF4) led to improved tolerance to salt and drought stresses in Arabidopsis. It also significantly affected the growth and development of transgenic plants. We detected that salt-induced expressions of a transcriptional repressor gene, AtERF4, and some Ser/Thr protein phosphatase2C genes, ABI1, ABI2 and AtPP2CA, were suppressed in BrERF4-overexpressing Arabidopsis plants. Furthermore, BrERF4 was induced by treatment with ethylene or methyljasmonate, but not by abscisic acid or NaCl in B. rapa. These results suggest that BrERF4 is activated through a network of different signaling pathways in response to salinity and drought.