Rice Cells Research Articles

Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins.

Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency. We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures. Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme.

Effect of steam explosion on nutritional components, physicochemical and rheological properties of brown rice powder.

Brown rice powder is underutilized mainly due to its lower starch digestibility and poor processing performance. The present study investigated the potential of steam explosion on the improvement of nutritional and physicochemical characteristic in brown rice powder and rheological property of paste. Compared with native brown rice powder, steam explosion at 0.5 MPa for 7 min increased the water-extractable arabinoxylans (5.77%), reducing sugar content (21.04%), and iodine blue value (30.38%), which indicated steam explosion that destroyed the intact cells of brown rice. Later the crystalline structure of brown rice powder was destroyed into an amorphous structure by steam explosion. Steam explosion enhanced the degree of gelatinization (4.76~351.85%) and solvent retention capacity (SRC) of brown rice powder, compared with native sample. The effect on the intact cells and starch structure of brown rice caused the starch digestibility enhancement remarkable. Viscoelastic profiles confirmed that steam explosion weakened the paste strength and elasticity corresponded with hardness and cohesiveness by increasing the loss factor (tanδ). This work provided important information for brown rice powder modified by steam explosion (0.5 MPa, 7 min) with good nutritional property (nutrients and digestibility) and processability (SRC, textural, and rheological property). Steam exploded brown rice powder (0.5 MPa, 7 min) could serve as a potential ingredient widely used in food products.

The Cd sequestration effects of rice roots affected by different Si management in Cd-contaminated paddy soil

The application of exogenous silicon (Si) reportedly is one of the eco-friendly practices to mitigate cadmium (Cd) phytotoxicity and regulate the chemical behaviors of Cd in the soil-rice system. But the efficiency of Si on the Cd retention by rice root varies with the Si fertilizer management. The objective of this paper was to interpret the differences in Cd immobilization by rice roots and relevant mechanisms under different ways of Si application (T-Si, supplied at transplanting stage; TJ-Si, split at transplanting and jointing stage with the ratio of 50 % to 50 %; J-Si, supplied at jointing stage and CK, none of Si application) in Cd-contaminated paddy soils. The results showed that the Cd-retention capacity of rice root was increased by 0.60 % ~ 3.06 % under different Si management when compared to CK. The concentrations of monosilicic acid in soils and in apoplast and symplast of roots were increased significantly by Si application, while Cd concentrations in apoplast and symplast of root were decreased by 28.50 % (T-Si), 40.64 % (TJ-Si) and 30.26 % (J-Si), respectively. The distribution of Cd in rice cell wall was increased significantly by TJ-Si. The Cd concentrations of inert fractions (F3, F4 and F6) in root of TJ-Si were raised obviously. Si application downregulated the expression of OsIRT2 and OsNramp5 while upregulated OsHMA3, and the expression of OsHMA3 treated by TJ-Si was obviously higher than CK and J-Si. The distributions of the passive Cd in roots bound with thiol compounds (NPT, GSH and PCs) and polysaccharide components (pectin, hemicelluloses 1 and hemicellulose 2) were raised much more by TJ-Si than by T-Si and J-Si. On the whole, compared with T-Si and J-Si, TJ-Si could more easily replenish soil available Si and enhance Cd sequestration in roots as the result of the decrease of Cd transport factor in roots. This study unravels some mechanisms about different Si management on increasing Cd retention and decreasing Cd migration in rice roots, and TJ-Si is worthy of being recommended.

Carbon-nanotube for Transient Expression in Rice Calli

Transient gene expression is an important technique in gene functional analysis, protein production and in plants. However, traditional transient expression methods using Agrobacterium are time-consuming with low efficiency. In this study, we demonstrated the use of a single-walled carbon nanotube (SWCNT) to deliver 35S:mCherry:pCXSN plasmid into rice calli. This transient expression protocol used a plastic medical syringe to create the physical pressure to help the delivery of plasmid DNA into plant cells. This protocol is relatively easy to perform and low cost. The transient expression was observed under fluorescence microscopy, and the mCherry fluorescence signal was quantified. The plasmid DNA was delivered into the rice cell using a 3:1 ratio (plasmid: carbon nanotube). The result showed that the mCherry signal of carbon nanotube + plasmid DNA treatment was the highest signal at 3 days post-transformation and decreased to a lower signal at 6 days post-transformation. Moreover, the quantitative analysis of mCherry mean intensity revealed that the signal intensity of carbon nanotube + plasmid DNA treatment was the highest level, and significantly higher than the control treatments at 3 days post-transformation. Plasmid DNA can be transported easily into plant calli using this carbon nanotube transient expression protocol.

A pH-responsive/sustained release nitrogen fertilizer hydrogel based on aminated cellulose nanofiber/cationic copolymer for application in irrigated neutral soils

Stimuli-responsive release nitrogen (N)-fertilizer hydrogels are new materials recently introduced to improve control and sustained release N-fertilizer hydrogels efficacy by adding additional functionality for greater N use efficiency. However, the stimuli-responsive behavior and release mechanisms of these fertilizer hydrogels in response to irrigated-soil stimulus fluctuations during different plant growth stages are still facing limitations and have not been considered. Herein, a novel N-fertilizer nanocomposite hydrogel with the function of N pH-responsive/sustained release (pHRSRNFH) was prepared based on wheat straw aminated-cellulose nanofibers (A-CNFs) and cationic poly(acrylamide-co-2-aminoethyl methacrylate hydrochloride) (PAM-PAEM) by direct ammonium nitrate (AN) fertilizer encapsulation. The fertilizer hydrogel structure, properties, and effects on N use efficiency and N metabolism process were characterized by FTIR, zeta potential, SEM, porosity, swelling-release behaviors, TEM, flowcytometric analysis, and N use efficiency indicators. The hydrogel nanocomposite with 3 wt% A-CNFs and 4 wt% AEM (pHRSRNFH4) exhibited desirable enhancement of characterizations, pH-responsive/sustained behaviors, and biodegradability. The soil and buffers release kinetic studies in-vitro revealed the best pH-dependent/sustained AN release of 3.00 and 2.69 mg.day−1 at pH 5.5 and 0.92 and 0.55 mg.day−1 at pH 7.4 from 1g pHRSRNFH4 for 58 and 65 days, respectively, desirably synchronizing with the pH fluctuations of irrigated-neutral soil. The whole lifetime of AN release data revealed that the three sustained delivery stages were governed by the First-order mechanism, followed by Zeroth-order, and finally via the Higuchi mechanism, covering the N crop requirements during different plant growth phases. Application of pHRSRNFH4 at 1 g kg−1 irrigated-neutral soil successfully improved water-holding capacity and N recovery efficiency by 15.7% and 38.6%, respectively, compared with free AN treated soil with over 90-days sustained AN release. Furthermore, positive impacts on rice growth indicators, N-nutrition status, and cell progression were achieved with a feasible-compatible application, demonstrating its further significant potential to be a viable alternative fertilizer formulation for N/water management of cropping systems in the irrigated-neutral soils.

Co-evolved plant and blast fungus ascorbate oxidases orchestrate the redox state of host apoplast to modulate rice immunity

Apoplastic ascorbate oxidases (AOs) play a critical role in reactive oxygen species (ROS)-mediated innate host immunity by regulating the apoplast redox state. To date, little is known about how apoplastic effectors of the rice blast fungus Magnaporthe oryzae modulate the apoplast redox state of rice to subvert plant immunity. In this study, we demonstrated that M. oryzae MoAo1 is an AO that plays a role in virulence by modulating the apoplast redox status of rice cells. We showed that MoAo1 inhibits the activity of rice OsAO3 and OsAO4, which also regulate the apoplast redox status and plant immunity. In addition, we found that MoAo1, OsAO3, and OsAO4 all exhibit polymorphic variations whose varied interactions orchestrate pathogen virulence and rice immunity. Taken together, our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state and its regulation in plant-pathogen interactions.

An amino acid transporter-like protein (OsATL15) facilitates the systematic distribution of thiamethoxam in rice for controlling the brown planthopper.

SummaryCharacterization and genetic engineering of plant transporters involved in the pesticide uptake and translocation facilitate pesticide relocation to the tissue where the pests feed, thus improving the bioavailability of the agrichemicals. We aimed to identify thiamethoxam (THX) transporters in rice and modify their expression for better brown planthopper (BPH) control with less pesticide application. A yeast library expressing 1385 rice transporters was screened, leading to the identification of an amino acid transporter‐like (ATL) gene, namely OsATL15, which facilitates THX uptake in both yeast cells and rice seedlings. In contrast to a decrease in THX content in osatl15 knockout mutants, ectopic expression of OsATL15 under the control of the CaMV 35S promoter or a vascular‐bundle‐specific promoter gdcsPpro significantly increased THX accumulation in rice plants, thus further enhancing the THX efficacy against BPH. OsATL15 was localized in rice cell membrane and abundant in the root transverse sections, vascular bundles of leaf blade, and stem longitudinal sections, but not in hull and brown rice at filling stages. Our study shows that OsATL15 plays an essential role in THX uptake and its systemic distribution in rice. OsATL15 could be valuable in achieving precise pest control by biotechnology approaches.

Transcriptional regulation of rice HSP101 promoter: Mitogen‐activated protein kinase‐mediated HSFA6a phosphorylation affects its stability and transactivation

AbstractHeat shock proteins (HSPs) and heat stress factors (HSFs) control the plant heat stress response to a large extent. HSP101 plays a decisive role in development of plant heat tolerance. We have previously shown that rice (Oryza sativa) cells contain 25 HSFs and among these, HSFA6a most predominantly binds to the HSP101 promoter and controls its transcript expression. This study shows that mitogen‐activated protein kinases (MAPKs), specifically MPK3, MPK4, and MPK6 phosphorylate HSFA6a. HSFA6a showed physical interaction with MPK3/MPK4, specifically in the nucleus and this interaction involved the C‐terminal end of HSFA6a. Four serine residues at positions Ser136, Ser141, Ser264, and Ser356 of HSFA6a are the putative sites of MAPK phosphorylation: we generated phospho‐mutant of HSFA6a by changing the serine residues to alanine either individually or all four together. The Hsp101 promoter binding potential of Ser136 mutant was enhanced while it declined for the other three phospho‐mutant HSFA6a forms. HSFA6a mutant lacking all the above four Ser residues exhibited reduced DNA binding and transactivation potential. We implicate the role of phosphorylation in the regulation of HSFA6a activity.

Developmental Differences between Anthers of Diploid and Autotetraploid Rice at Meiosis.

Newly synthetic autotetraploid rice shows lower pollen fertility and seed setting rate relative to diploid rice, which hinders its domestication and breeding. In this study, cytological analysis showed that at meiosis I stage, an unbalanced segregation of homologous chromosomes, occurred as well as an early degeneration of tapetal cells in autotetraploid rice. We identified 941 differentially expressed proteins (DEPs) in anthers (meiosis I), including 489 upregulated and 452 downregulated proteins. The DEPs identified were related to post-translational modifications such as protein ubiquitination. These modifications are related to chromatin remodeling and homologous recombination abnormalities during meiosis. In addition, proteins related to the pentose phosphate pathway (BGIOSGA016558, BGIOSGA022166, and BGIOSGA028743) were downregulated. This may be related to the failure of autotetraploid rice to provide the energy needed for cell development after polyploidization, which then ultimately leads to the early degradation of the tapetum. Moreover, we also found that proteins (BGIOSGA017346 and BGIOSGA027368) related to glutenin degradation were upregulated, indicating that a large loss of glutenin cannot provide nutrition for the development of tapetum, resulting in early degradation of tapetum. Taken together, these evidences may help to understand the differences in anther development between diploid and autotetraploid rice during meiosis.

Fluorescent cytoskeletal markers reveal associations between the actin and microtubule cytoskeleton in rice cells.

Rice (Oryza sativa) is one of our main food crops, feeding ∼3.5 billion people worldwide. An increasing number of studies note the importance of the cytoskeleton, including actin filaments and microtubules, in rice development and environmental responses. Yet, reliable in vivo cytoskeleton markers are lacking in rice, which limits our knowledge of cytoskeletal functions in living cells. Therefore, we generated bright fluorescent marker lines of the actin and microtubule cytoskeletons in rice, suitable for live-cell imaging in a wide variety of rice tissues. Using these lines, we show that actin bundles and microtubules engage and co-function during pollen grain development, how the cytoskeletal components are coordinated during root cell development, and that the actin cytoskeleton is robust and facilitates microtubule responses during salt stress. Hence, we conclude that our cytoskeletal marker lines, highlighted by our findings of cytoskeletal associations and dynamics, will substantially further future investigations in rice biology.

Multiplex precision gene editing by a surrogate prime editor in rice

Multiplex precision gene editing by a surrogate prime editor in rice

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method.

Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5′–3′ backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.

Two Magnaporthe appressoria-specific (MAS) proteins, MoMas3 and MoMas5, are required for suppressing host innate immunity and promoting biotrophic growth in rice cells.

In the devastating rice blast fungus Magnaporthe oryzae, six Magnaporthe appressoria‐specific (MAS) proteins are encoded by MoGAS1, MoGAS2 and MoMAS3–MoMAS6. MoGAS1 and MoGAS2 were previously characterized as M. oryzae virulence factors; however, the roles of the other four genes are unknown. Here, we found that, although the loss of any MAS gene did not affect appressorial formation or vegetative growth, ∆Momas3 and ∆Momas5 mutant strains (but not the others) were reduced in virulence on susceptible CO‐39 rice seedlings. Focusing on ∆Momas3 and ∆Momas5 mutant strains, we found that they could penetrate host leaf surfaces and fill the first infected rice cell but did not spread readily to neighbouring cells, suggesting they were impaired for biotrophic growth. Live‐cell imaging of fluorescently labelled MoMas3 and MoMas5 proteins showed that during biotrophy, MoMas3 localized to the apoplastic compartment formed between fungal invasive hyphae and the plant‐derived extra‐invasive hyphal membrane while MoMas5 localized to the appressoria and the penetration peg. The loss of either MoMAS3 or MoMAS5 resulted in the accumulation of reactive oxygen species (ROS) in infected rice cells, resulting in the triggering of plant defences that inhibited mutant growth in planta. ∆Momas3 and ∆Momas5 biotrophic growth could be remediated by inhibiting host NADPH oxidases and suppressing ROS accumulation. Thus, MoMas3 and MoMas5 are novel virulence factors involved in suppressing host plant innate immunity to promote biotrophic growth.

Rice sl-MH-1 mutant induces cell death and confers blast resistance via the synergistic roles of signaling systems

Serotonin is ubiquitous across all forms of life and functions in responses to biotic and abiotic stresses. In rice, the conversion of tryptamine to serotonin is catalyzed by Sekiguchi lesion (SL). Previous studies have identified an sl mutation (a null mutation of SL) in several rice varieties and confirmed its increase of resistance and cell death. However, a systematic understanding of the reprogrammed cellular processes causing cell death and resistance is lacking. We performed a multi-omics analysis to clarify the fundamental mechanisms at the protein, gene transcript, and metabolite levels. We found that cell death and Magnaporthe oryzae (M. oryzae) infection of the sl-MH-1 mutant activated plant hormone signal transduction involving salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in multiple regulatory layers. We characterized the dynamic changes of several key hormone levels during disease progression and under the cell death conditions and showed that SA and JA positively regulated rice cell death and disease resistance. SL-overexpressing lines confirmed that the sl-MH-1 mutant positively regulated rice resistance to M. oryzae. Our studies shed light on cell death and facilitate further mechanistic dissection of programmed cell death in rice.

Expanding the targeting scope of FokI‐dCas nuclease systems with SpRY and Mb2Cas12a

CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system wasdeveloped that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system wasdeveloped that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.

Identification of Differentially Expressed Genes Reveal Conserved Mechanisms in the Rice-Magnaporthe oryzae Interaction

Magnaporthe oryzae causes rice blast disease and is responsible for major losses in rice production worldwide. Although numerous studies have focused on the interactions between Oryza sativa and M. oryzae, to date, the conserved mechanisms remain in part unclear. In this study, a comparative analysis of transcriptomes of O. sativa L. ssp. japonica cv. ‘Nipponbare’ interacting with three M. oryzae strains (248, 235, and 163) were performed to explore the conserved molecular mechanisms. Differentially expressed genes with similar expression patterns in the interactions between cultivar ‘Nipponbare’ and three M. oryzae strains were defined as Conserved Differentially Expressed Genes (CDEGs). These included 3,647 O. sativa CDEGs and 3,655 M. oryzae CDEGs. Four rice CDEGs (LOC_Os03g19270, LOC_Os07g36600, LOC_Os05g28740, and LOC_Os01g32780) encoding universal stress protein (USP) were induced within 24 h post-inoculation (hpi) by three M. oryzae strains. Meanwhile, overexpression of LOC_Os07g36600 resulted in enhanced rice resistance against M. oryzae. Furthermore, four rice genes coding light-harvesting chlorophyll a/b-binding (LHC) protein (LOC_Os02g52650, LOC_Os09g12540, LOC_Os11g13850, LOC_Os05g22730) were also identified as CDEGs and were induced at 48 hpi, which might contribute to blast resistance through reactive oxygen species (ROS) accumulation. MoCDIP4 is M. oryzae effector inducing rice cell death and were verified that include AA9 CAZy domain (namely GH61 domain). In this study, we found seven MoCDIP4-homologous genes coding proteins with signal peptides and AA9 CAZy domains, which were continuously up-regulated across all infection stages relative to uninoculated control. This study uncovered that genes are required for conserved mechanisms of rice-M. oryzae interaction, which includes rice genes encoding USP proteins and LHC proteins, as well as M. oryzae genes encoding AA9 proteins. This study will help us to understand how O. sativa responds to M. oryzae infections and the molecular mechanisms of M. oryzae pathogenicity.

Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture

High-mannose glycans, containing 6–9 mannose residues, are favorable for in vitro mannose phosphorylation to produce mannose-6-phosphate (M6P) residues, which are essential for uptake into target cells during enzyme replacement therapy of Pompe disease. Golgi α-mannosidase-I mediates mannose trimming in the N-glycosylation pathway. In this study, mutant rice with a T-DNA insertion in the Os04g0606400 (∆α-manI) gene was used to produce recombinant human acid α-glucosidase (rhGAA). Expressed and secreted rhGAA from ∆α-manI (∆α-manI-GAA) in suspension culture media was purified, and analysed by MALDI-TOF. It showed the presence of high-mannose type N-glycans with Man8 (63.8%), Man7 (22.5%), and Man6 (5.4%).

Production and antioxidant activity of secondary metabolites in Hassawi rice (Oryza sativa L.) cell suspension under salicylic acid, yeast extract, and pectin elicitation

Production and antioxidant activity of secondary metabolites in Hassawi rice (Oryza sativa L.) cell suspension under salicylic acid, yeast extract, and pectin elicitation

Enhancing Antioxidant Activities and Anti-Aging Effect of Rice Stem Cell Extracts by Plasma Treatment

Plant-derived substances exhibit antioxidant and antibacterial activities and have been proven to have beneficial effects in wound healing and skin regeneration. Plant stem cells have recently received much attention as research materials in cosmetic development because they promote regeneration after damage. In this paper, we demonstrate for the first time that the plasma treatment of stem cells obtained from rice-seed embryos can be effective in enhancing antioxidant activity and in regenerating human skin. We investigated this potential utilizing micro-DBD (Dielectric Barrier Discharge) plasma as a pretreatment technique to enhance the vitality and functional activity of rice stem cells. The results of the cell culture experiments show that plasma-treated rice stem cell extracts (RSCE) have promising antioxidant and anti-skin aging activities. The results of quantitative real-time PCR (qRT-PCR) for major antioxidant enzymes and anti-aging genes confirm that the plasma technique used in the pretreatment of RSCE was able to enhance cell activities in skin regeneration, including cell survival, proliferation, and collagen enhancement for Human Fibroblast (HFB) degraded by oxidative stress. These results show that the relatively low energy of less than 300 W and an amount of NOx-based reactive nitrogen species (RNS) from plasma discharge of about 3 μL/L were the key factors and that RSCE, of which the antioxidant activity was enhanced by plasma treatment, appeared to be a major contributor to the protective effect of HFB against oxidative stress. Plasma-treated RSCE induced excellent anti-aging properties by stimulating HFB to promote collagen synthesis, thereby promoting skin regeneration. These properties can protect the skin from various oxidative stresses. This study demonstrates that plasma-treated extracts of stem cells derived from rice-seed embryos have an excellent regenerative effect on aging-treated HFB. Our results demonstrate the potential utility of plasma-treated RSCE as a skin anti-aging agent in cosmeceutical formulations for the first time.

Function of hydroxycinnamoyl transferases for the biosynthesis of phenolamides in rice resistance to Magnaporthe oryzae

Phenolamide (PA) metabolites play important roles in the interaction between plants and pathogens. The putrescine hydroxycinnamoyl transferase genes OsPHT3 and OsPHT4 positively regulate rice cell death and resistance to Magnaporthe oryzae. The bZIP transcription factor APIP5, a negative regulator of cell death and rice immunity, directly binds to the OsPHT4 promoter to regulate putrescine-derived PAs. Whether other hydroxycinnamoyl transferase (HT) genes also participate in APIP5-mediated immunity remains unclear. Surprisingly, we find that genes encoding agmatine hydroxycinnamoyl transferases OsAHT1 and OsAHT2, tryptamine hydroxycinnamoyl transferases OsTBT1 and OsTBT2, and tyramine hydroxycinnamoyl transferases OsTHT1 and OsTHT2, responsible for the biosynthesis of polyamine-derived PAs are all up-regulated in APIP5-RNAi transgenic plants compared with segregated wild-type rice. Furthermore, both OsAHT1/2 and OsTBT1/2 are induced during M. oryzae infection, showing expression patterns similar to those previously reported for OsTHT1/2 and OsPHT3/4. Transgenic plants overexpressing either OsAHT2-GFP or OsTBT1-GFP show enhanced resistance against M. oryzae and accumulated more PA metabolites and lignin compared with wild-type plants. Interestingly, as demonstrated for OsPHT4, APIP5 directly binds to the promoters of OsAHT1/2, OsTBT1/2, and OsTHT1/2, repressing their transcription. Together, these results indicate that the HT genes are common targets of APIP5 and that PAs play critical roles in rice immunity.