Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Ectopic expression of a thaumatin-like protein impairs the timely deposition and dissolution of callose during microsporogenesis, leading to microspore death and male sterility in Arabidopsis.

KEY MESSAGE: Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Protoplast isolation and transient gene expression in different petunia cultivars.

The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 105 protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.

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.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

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.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

A Raf-like kinase is required for smoke-induced seed dormancy in Arabidopsis thaliana.

Plants sense and integrate diverse stimuli to determine the timing for germination. A smoke compound, 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide, TMB), has been identified to inhibit the seed germination of higher plants. To understand the mode of action, we examined various physiological and molecular aspects of the TMB-dependent inhibition of seed germination in Arabidopsis thaliana The results indicated that the effect of TMB is due to the enhanced physiological dormancy, which is modulated by other dormancy regulatory cues such as after-ripening, stratification, and ABA/GA signaling. In addition, gene expression profiling showed that TMB caused genome-wide transcriptional changes, altering the expression of a series of dormancy-related genes. Based on the TMB-responsive physiological contexts in Arabidopsis, we performed mutant screening to isolate genetic components that underpin the TMB-induced seed dormancy. As a result, the TMB-RESISTANT1 (TES1) gene in Arabidopsis, encoding a B2 group Raf-like kinase, was identified. Phenotypic analysis of the tes1 mutant implicated that TES1 has a critical role in the TMB-responsive gene expression and the inhibition of seed germination. Taken together, we propose that plants have been equipped with a TMB sensory pathway through which the TMB induces the seed dormancy in a TES1-dependent way.

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3.

Pollen in angiosperms plays a critical role in double fertilization by germinating and elongating pollen tubes rapidly in one direction to deliver sperm. In this process, the secretory vesicles deliver cell wall and plasma membrane materials, and excessive materials are sequestered via endocytosis. However, endocytosis in plants is poorly understood. AP180 N-terminal homology (ANTH) domain-containing proteins function as adaptive regulators for clathrin-mediated endocytosis in eukaryotic systems. Here, we identified 17 ANTH domain-containing proteins from rice based on a genome-wide investigation. Motif and phylogenomic analyses revealed seven asparagine-proline-phenylalanine (NPF)-rich and 10 NPF-less subgroups of these proteins, as well as various clathrin-mediated endocytosis-related motifs in their C-terminals. To investigate their roles in pollen germination, we performed meta-expression analysis of all genes encoding ANTH domain-containing proteins in Oryza sativa (OsANTH genes) in anatomical samples, including pollen, and identified five mature pollen-preferred OsANTH genes. The subcellular localization of four OsANTH proteins that were preferentially expressed in mature pollen can be consistent with their role in endocytosis in the plasma membrane. Of them, OsANTH3 represented the highest expression in mature pollen. Functional characterization of OsANTH3 using T-DNA insertional knockout and gene-edited mutants revealed that a mutation in OsANTH3 decreased seed fertility by reducing the pollen germination percentage in rice. Thus, our study suggests OsANTH3-mediated endocytosis is important for rice pollen germination.

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.

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen-specific gene mutants in rice (Oryza sativa), we identified a T-DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one-to-one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven-WD40-motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori-2, created through CRISPR-Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori-2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen-specific genes were downregulated in gori-2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo-/exocytosis complexes that could mediate pollen tube growth in rice.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

OsbHLH073 Negatively Regulates Internode Elongation and Plant Height by Modulating GA Homeostasis in Rice.

Internode elongation is one of the key agronomic traits determining a plant's height and biomass. However, our understanding of the molecular mechanisms controlling internode elongation is still limited in crop plant species. Here, we report the functional identification of an atypical basic helix-loop-helix transcription factor (OsbHLH073) through gain-of-function studies using overexpression (OsbHLH073-OX) and activation tagging (osbhlh073-D) lines of rice. The expression of OsbHLH073 was significantly increased in the osbhlh073-D line. The phenotype of osbhlh073-D showed semi-dwarfism due to deficient elongation of the first internode and poor panicle exsertion. Transgenic lines overexpressing OsbHLH073 confirmed the phenotype of the osbhlh073-D line. Exogenous gibberellic acid (GA3) treatment recovered the semi-dwarf phenotype of osbhlh073-D plants at the seedling stage. In addition, quantitative expression analysis of genes involving in GA biosynthetic and signaling pathway revealed that the transcripts of rice ent-kaurene oxidases 1 and 2 (OsKO1 and OsKO2) encoding the GA biosynthetic enzyme were significantly downregulated in osbhlh073-D and OsbHLH073-OX lines. Yeast two-hybrid and localization assays showed that the OsbHLH073 protein is a nuclear localized-transcriptional activator. We report that OsbHLH073 participates in regulating plant height, internode elongation, and panicle exsertion by regulating GA biosynthesis associated with the OsKO1 and OsKO2 genes.

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.

In vitro propagation method for production of morphologically and genetically stable plants of different strawberry cultivars.

BACKGROUND: As strawberries are susceptible to somaclonal variation when propagated by tissue culture techniques, it is challenging to obtain the true-to-type plants necessary for continuous production of fruits of stable quality. Therefore, we aimed to develop an in vitro propagation method for the production of true-to-type plants of five different strawberry cultivars from meristems cultured in media containing different concentrations of kinetin (Kn). RESULTS: For all the cultivars, shoot induction was successful only in the meristems cultured in the medium without Kn and the medium containing 0.5 mg L-1 Kn. The shoots obtained from explants cultured in media supplemented with 0.5 mg L-1 Kn exhibited better plant growth parameters than those cultured in media without Kn and were genetically stable when compared with conventionally propagated plants for all the cultivars. Vegetative and sexual characters and fruit quality attributes observed in the plants derived from meristems cultured on 0.5 mg L-1 Kn and the conventionally propagated plants were not significantly different when grown for three continuous growing seasons under greenhouse conditions. CONCLUSION: The culture of meristems in the medium containing 0.5 mg L-1 Kn is suitable for the efficient propagation of true-to-type plants of different strawberry cultivars and continuous production of fruits with stable quality. Hence, we expect that the method presented in this study will be helpful for the commercial production of true-to-type plants generated in vitro for other strawberry cultivars.

PACLOBUTRAZOL-RESISTANCE Gene Family Regulates Floral Organ Growth with Unequal Genetic Redundancy in Arabidopsis thaliana.

A PACLOBUTRAZOL-RESISTANCE (PRE) gene family, consisting of six genes in Arabidopsis thaliana, encodes a group of helix-loop-helix proteins that act in the growth-promoting transcriptional network. To delineate the specific role of each of the PRE genes in organ growth, we took a reverse genetic approach by constructing high order pre loss-of-function mutants of Arabidopsis thaliana. In addition to dwarf vegetative growth, some double or high order pre mutants exhibited defective floral development, resulting in reduced fertility. While pre2pre5 is normally fertile, both pre2pre6 and pre5pre6 showed reduced fertility. Further, the reduced fertility was exacerbated in the pre2pre5pre6 mutant, indicative of the redundant and critical roles of these PREs. Self-pollination assay and scanning electron microscopy analysis showed that the sterility of pre2pre5pre6 was mainly ascribed to the reduced cell elongation of anther filament, limiting access of pollens to stigma. We found that the expression of a subset of flower-development related genes including ARGOS, IAA19, ACS8, and MYB24 was downregulated in the pre2pre5pre6 flowers. Given these results, we propose that PREs, with unequal functional redundancy, take part in the coordinated growth of floral organs, contributing to successful autogamous reproduction in Arabidopsis thaliana.

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.