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Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

Plants are sessile organisms that have developed hydrophobic cuticles that cover their aerial epidermal cells to protect them from terrestrial stresses. The cuticle layer is mainly composed of cutin, a polyester of hydroxy and epoxy fatty acids, and cuticular wax, a mixture of very-long-chain fatty acids (>20 carbon atoms) and their derivatives, aldehydes, alkanes, ketones, alcohols, and wax esters. During the last 30 years, forward and reverse genetic, transcriptomic, and biochemical approaches have enabled the identification of key enzymes, transporters, and regulators involved in the biosynthesis of cutin and cuticular waxes. In particular, cuticular wax biosynthesis is significantly influenced in an organ-specific manner or by environmental conditions, and is controlled using a variety of regulators. Recent studies on the regulatory mechanisms underlying cuticular wax biosynthesis have enabled us to understand how plants finely control carbon metabolic pathways to balance between optimal growth and development and defense against abiotic and biotic stresses. In this review, we summarize the regulatory mechanisms underlying cuticular wax biosynthesis at the transcriptional, post-transcriptional, post-translational, and epigenetic levels.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1–84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1–9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition.

Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

The CRISPR–Cas9 system is a powerful tool for editing genes of interest in specific plant genomes. Indeed, genome-editing systems have been used to enhance a variety of agricultural traits and to study gene functions in many plant species, and the plasmid-mediated delivery of Cas9 and single guide RNA (sgRNA) to plants has been reported to facilitate highly efficient editing. However, the random and stable integration of plasmid DNA sequences into plant genomes can cause insertional mutagenesis, and an additional step is required to remove such foreign sequences from edited plant genomes. Accordingly, the aim of the present study was to investigate the effectiveness of directly delivering purified CRISPR–Cas9 ribonucleoproteins (RNPs) to protoplasts from cabbage (Brassica oleracea var. capitata), an important cruciferous vegetable. The flowering-time regulator gene GIGANTEA (GI) was targeted, with the goal of delaying flowering time and prolonging vegetative growth. We investigated the targeted mutagenesis insertion and deletion rates using targeted deep sequencing. The mutation frequency achieved using one of the sgRNAs (sgRNA2) was 2% in the infected protoplast. The shoots were regenerated from 44% (46/103) of protoplast-derived calli. Consequently, three independent and completely transgene-free mutants were obtained, including one homogeneous biallelic line in which both GI alleles were successfully edited, thereby yielding a complete GI knockout line. These results suggest that the transgene-free CRISPR–Cas9 system is a promising tool for improving agricultural beneficial traits of cabbage.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3'H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-&beta, cyclodextrin (Me&beta, CD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 &mu, M MJ on cells. We also found that the combined treatment of cells for seven days with MJ and Me&beta, CD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion, however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows Me&beta, CD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

The hydrophobic biopolymer suberin, which is deposited in the root endodermis and seed coats, functions as an extracellular barrier against uncontrolled water, gas, and ion loss. Suberin monomers synthesized in the endoplasmic reticulum (ER) are exported through the plasma membrane to the apoplast. However, limited information is available about the molecular mechanisms underlying suberin monomer export and assembly. In this study, we investigated the in planta role of LTPG15 encoding a glycosylphosphatidylinositol (GPI)-anchored lipid transfer protein. LTPG15 was predominantly expressed in the root endodermis and seed coat. Fluorescent signals from LTPG15:eYFP were detected in the plasma membrane in tobacco epidermis. Disruption of LTPG15 caused a significant decrease in the levels of fatty acids (C20-C24), primary alcohols (C20 and C22), ω-hydroxy fatty acids (C22 and C24), and α,ω-alkanediols (C20 and C22), but an increase in the amounts of primary alcohols and hydroxy fatty acids with C16 and C18 in seed coats. The mutant phenotype was restored to that of the wild type (WT) by the expression of LTPG15 driven by its own promoter. Seed coats of ltpg15 had an increase in permeability to tetrazolium salts compared with WT seed coats. ltpg15 seeds were more sensitive than WT seeds to inhibition of germination and seedling establishment by salt and osmotic stress treatments. Taken together, our results indicate that LTPG15 is involved in suberin monomer export in seed coats, and this highlights the role of Type G non-specific lipid transfer proteins (LTPGs) in very-long-chain fatty acids and their derivatives' export for suberin polyester formation.

During the evolution of land plants from aquatic to terrestrial environments, their aerial surfaces were surrounded by cuticle composed of cutin and cuticular waxes to protect them from environmental stresses. Glycerol-3-phosphate acyltransferase (GPAT) harboring bifunctional sn-2 acyltransferase/phosphatase activity produces 2-monoacylglycerol, a precursor for cutin synthesis. Here, we report that bifunctional sn-2 GPATs play roles in cuticle biosynthesis and gametophore development of Physcomitrella patens. Land plant-type cuticle was observed in gametophores but not in protonema. The expression of endoplasmic reticulum-localized PpGPATs was significantly upregulated in gametophores compared with protonema. Floral organ fusion and permeable cuticle phenotypes of Arabidopsis gpat6-2 petals were rescued to the wild type (WT) by the expression of PpGPAT2 or PpGPAT4. Disruption of PpGPAT2 and PpGPAT4 caused a significant reduction of total cutin loads, and a prominent decrease in the levels of palmitic and 10,16-dihydroxydecanoic acids, which are major cutin monomers in gametophores. Δppgpat2 mutants displayed growth retardation, delayed gametophore development, increased cuticular permeability, and reduced tolerance to drought, osmotic and salt stresses compared to the WT. Genome-wide analysis of genes encoding acyltransferase or phosphatase domains suggested that the occurrence of sn-2 GPATs with both domains may be a key event in cuticle biogenesis of land plants.

The importance of the cuticular layer in regulating a plant’s water status and providing protection from environmental challenges has been recognized for a long time. The cuticular layer in plants restricts non-stomatal water loss and protects plants against damage from pathogen infection and UV radiation. Much genetic and biochemical research has been done about cutin and wax transportation in Arabidopsis thaliana, but little is known about it in rice. Here, we report that a rice ATP-binding cassette (ABC) transporter, OsABCG9, is essential for normal development during vegetative growth and could play a critical role in the transportation of epicuticular wax in rice. Rice phenotypes with mutated OsABCG9 exhibited growth retardation and sensitivity to low humidity. The total amount of cuticular wax on the leaves of the osabcg9-1 mutant diminished by 53% compared with the wild type, and wax crystals disappeared completely in osabcg9-2 mutant leaves. However, OsABCG9 does not seem to be involved in cutin transportation, even though its ortholog in Arabidopsis, AtABCG11, transports both wax and cutin. Furthermore, the osabcg9-1 mutant had increased leaf chlorophyll leaching and more severe drought susceptibility. This study provides new insights about differences between rice and A. thaliana in wax and cutin transportation associated with the ABCG family during evolution.

Aerial plant surfaces are coated by a cuticular wax layer to protect against environmental stresses, such as desiccation. In this study, we investigated the functional relationship between MYB94 and MYB96 transcription factors involved in cuticular wax biosynthesis. Both MYB94 and MYB96 transcripts were abundantly expressed in the aerial organs of Arabidopsis, and significantly induced at the same or similar time points under conditions of drought. MYB94 complemented the wax-deficient phenotype of the myb96 loss-of-function mutant under both well-watered and drought stress conditions. The magnitude of decrease in total wax load in the myb94 myb96 double mutant was almost equal to the sum of the reduced wax loads in the individual myb94 and myb96 mutants under both conditions. Leaves of the myb94 myb96 mutant lost water through the cuticle faster than those of myb94 or myb96 plants. Transcript levels of wax biosynthetic genes were decreased in the single mutants, and further reduced in the double mutant, relative to the wild type, under drought and ABA treatment conditions. MYB94 and MYB96 interact with the same regions containing MYB consensus motifs in the promoter regions of wax biosynthetic genes. The data collectively indicate that MYB94 and MYB96 exert an additive effect on cuticular wax biosynthesis, which may represent an efficient adaptive mechanism of response to drought in plants.

The aerial parts of plants are covered with a cuticle, a hydrophobic layer consisting of cutin polyester and cuticular waxes that protects them from various environmental stresses. Cuticular waxes mainly comprise very long chain fatty acids and their derivatives such as aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax esters that are also important raw materials for the production of lubricants, adhesives, cosmetics, and biofuels. The major function of cuticular waxes is to control non-stomatal water loss and gas exchange. In recent years, the in planta roles of many genes involved in cuticular wax biosynthesis have been characterized not only from model organisms like Arabidopsis thaliana and saltwater cress (Eutrema salsugineum), but also crop plants including maize, rice, wheat, tomato, petunia, Medicago sativa, Medicago truncatula, rapeseed, and Camelina sativa through genetic, biochemical, molecular, genomic, and cell biological approaches. In this review, we discuss recent advances in the understanding of the biological functions of genes involved in cuticular wax biosynthesis, transport, and regulation of wax deposition from Arabidopsis and crop species, provide information on cuticular wax amounts and composition in various organs of nine representative plant species, and suggest the important issues that need to be investigated in this field of study.