Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.

BACKGROUND: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas. RESULTS: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L- 1), kinetin (KN, 0, 0.5, and 1.0 mg L- 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L- 1 2,4-D + 0.5 mg L- 1 KN + 2 nM PSK, 0.5 mg L- 1 2,4-D + 0.9 mg L- 1 KN and 90 nM PSK, and 1.5 mg L- 1 2,4-D and 1 mg L- 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration. CONCLUSIONS: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.

Silicon enhances stem strength by promoting lignin accumulation in herbaceous peony (Paeonia lactiflora Pall.).

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-end cut flower, but stem bending caused by low stem strength severely decreases its quality. To enhance stem strength, the regulatory effects of exogenous silicon were investigated in P. lactiflora. The results showed that silicon application enhanced stem strength by increasing the thickness of secondary cell walls and the layers of thickened secondary cells. Moreover, more lignin accumulated, particularly G-lignin and S-lignin, and the activities of lignin biosynthetic enzymes increased with silicon application. In addition, based on transcriptome analysis, silicon application induced the expression of genes participating in lignin biosynthesis pathway. Among them, hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT1) was isolated from P. lactiflora and found to be mainly localized in the cytoplasm of cells. Overexpression of PlHCT1 increased the layers of thickened secondary cells and lignin accumulation in tobacco, resulting in enhanced stem strength and demonstrably straight stems. Finally, silicon content, lignin content and PlHCT1 expression in P. lactiflora cultivars with high stem strengths were totally higher than those in cultivars with low stem strengths. These results indicated that silicon application enhanced stem strength by promoting lignin accumulation in P. lactiflora, which has prospects for stem quality improvement in general.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Visualization and quantification of cadmium accumulation, chelation and antioxidation during the process of vacuolar compartmentalization in the hyperaccumulator plant Solanum nigrum L.

Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the proportion of vacuolar Cd in protoplasts showed its ultimate storage capacity (82.24 %-83.40 %), and the Cd concentration stored in the protoplast maintained at a certain level (73.81-77.46 mg L-1). Besides, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2 = 0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.

Effectors of Puccinia striiformis f. sp. tritici Suppressing the Pathogenic-Associated Molecular Pattern-Triggered Immune Response Were Screened by Transient Expression of Wheat Protoplasts.

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.

Enhanced polyethylene glycol (PEG)-mediated protoplast transformation system for the phytopathogenic fungus, Ganoderma boninense.

The basidiomycete fungus, Ganoderma boninense, has been identified as the main causal agent of oil palm basal stem rot (BSR) disease which has caused significant economic losses to the industry especially in Malaysia and Indonesia. Various efforts have been initiated to understand the disease and this plant pathogen especially at the molecular level. This is the first study of its kind on the development of a polyethylene glycol (PEG)-mediated protoplast transformation system for G. boninense. Based on the minimal inhibitory concentration study, 60 µg/mL and above of hygromycin were effective to completely inhibit G. boninense growth. Approximately 5.145 × 107 cells/mL of protoplasts with the viability of 97.24% was successfully obtained from G. boninense mycelium tissue. The PEG-mediated G. boninense protoplast transformation using 1 µg of transformation vector, 25% of PEG solution, 10 min of pre-transformation incubation, and 30 min of post-transformation incubation has improved the transformation rate as compared with the previous reported protocols for other basidiomycete fungi. Optimization of four transformation parameters has improved the transformation efficiency of G. boninense from an average of 2 to 67 putative transformants. The presence of hygromycin phosphotransferase (hpt) and enhanced green fluorescent protein (eGFP) genes in the putative transformants was detected by PCR and verified by gene sequence analysis. Southern hybridization result further confirmed the integration of hpt gene in G. boninense transformants, and the green fluorescent signal was detected in the G. boninense transformants under the microscopic analysis. The establishment of this transformation system will accelerate the gene function studies of G. boninense especially those genes that may contribute to the pathogenesis of this fungus in oil palm.

The calcium-dependent protein kinase ZmCDPK7 functions in heat-stress tolerance in maize.

Global warming poses a serious threat to crops. Calcium-dependent protein kinases (CDPKs)/CPKs play vital roles in plant stress responses, but their exact roles in plant thermotolerance remains elusive. Here, we explored the roles of heat-induced ZmCDPK7 in thermotolerance in maize. ZmCDPK7-overexpressing maize plants displayed higher thermotolerance, photosynthetic rates, and antioxidant enzyme activity but lower H2 O2 and malondialdehyde (MDA) contents than wild-type plants under heat stress. ZmCDPK7-knockdown plants displayed the opposite patterns. ZmCDPK7 is attached to the plasma membrane but can translocate to the cytosol under heat stress. ZmCDPK7 interacts with the small heat shock protein sHSP17.4, phosphorylates sHSP17.4 at Ser-44 and the respiratory burst oxidase homolog RBOHB at Ser-99, and upregulates their expression. Site-directed mutagenesis of sHSP17.4 to generate a Ser-44-Ala substitution reduced ZmCDPK7's enhancement of catalase activity but enhanced ZmCDPK7's suppression of MDA accumulation in heat-stressed maize protoplasts. sHSP17.4, ZmCDPK7, and RBOHB were less strongly upregulated in response to heat stress in the abscisic acid-deficient mutant vp5 versus the wild type. Pretreatment with an RBOH inhibitor suppressed sHSP17.4 and ZmCDPK7 expression. Therefore, abscisic acid-induced ZmCDPK7 functions both upstream and downstream of RBOH and participates in thermotolerance in maize by mediating the phosphorylation of sHSP17.4, which might be essential for its chaperone function.

STOP1 Regulates LKS1 Transcription and Coordinates K+/NH4+ Balance in Arabidopsis Response to Low-K+ Stress.

Potassium and nitrogen are essential mineral elements for plant growth and development. The protein kinase LKS1/CIPK23 is involved in both K+ and NH4+ uptake in Arabidopsis root. The transcripts of LKS1 can be induced by low K+ (0.1 mM) and high NH4+ (30 mM); however, the molecular mechanism is still unknown. In this study, we isolated the transcription factor STOP1 that positively regulates LKS1 transcription in Arabidopsis responses to both low-K+ and high-NH4+ stresses. STOP1 proteins can directly bind to the LKS1 promoter, promoting its transcription. The stop1 mutants displayed a leaf chlorosis phenotype similar to lks1 mutant when grown on low-K+ and high-NH4+ medium. On the other hand, STOP1 overexpressing plants exhibited a similar tolerant phenotype to LKS1 overexpressing plants. The transcript level of STOP1 was only upregulated by low K+ rather than high NH4+; however, the accumulation of STOP1 protein in the nucleus was required for the upregulation of LKS1 transcripts in both low-K+ and high-NH4+ responses. Our data demonstrate that STOP1 positively regulates LKS1 transcription under low-K+ and high-NH4+ conditions; therefore, LKS1 promotes K+ uptake and inhibits NH4+ uptake. The STOP1/LKS1 pathway plays crucial roles in K+ and NH4+ homeostasis, which coordinates potassium and nitrogen balance in plants in response to external fluctuating nutrient levels.

TaYS1A, a Yellow Stripe-Like Transporter Gene, Is Required for Wheat Resistance to Puccinia striiformis f. sp. Tritici.

Yellow stripe-like (YSL) transporters are required for the transportation of metal-phytosiderophores and are structurally related to metal-nicotianamine complexes. Some studies also reported the involvement of YSL transporters in pathogen-induced defense. However, the molecular mechanisms of YSL genes involved in biotic stress responses are still not clear, especially in cereal crops. This study aimed to functionally characterize TaYS1A during the interaction of wheat and Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust disease. TaYS1A was localized in the cell membrane of wheat protoplasts and Nicotiana benthamiana cells. TaYS1A was significantly up-regulated in wheat leaves after being infected with the avirulent Pst isolate CYR23 and after treatment with salicylic acid (SA). Silencing of TaYS1A by the virus-induced gene silencing method enhanced the susceptibility of wheat to Pst accompanied by reducing the accumulation of SA and H2O2 and down-regulating the transcriptions of TaPR1 and TaPR2. In addition, TaYS1A was found to interact with TaNH2, a homolog of OsNH2, by yeast-two-hybrid assay, and silencing of TaYS1A diminished the expression of TaNH2. Our findings suggested the existence of positive regulation of TaYS1A in providing resistance against Pst by modulating SA-induced signaling and offered new insight into the biological role of YSL in wheat against pathogens.

Multiple Xanthomonas campestris pv. campestris 8004 type III effectors inhibit immunity induced by flg22.

MAIN CONCLUSION: Xanthomonas campestris pv. campestris 8004 secretes several effector proteins that interfere with plant phosphorylation. Xanthomonas campestris pv. campestris (Xcc) can infect cruciferous plants and cause black rot. The strain Xcc8004 secretes effector proteins that interfere with plant cellular processes into host cells using a type III secretion (T3S) system. Several of the 24 predicted T3S effectors in the Xcc8004 genome have been implicated in the suppression of the Arabidopsis thaliana pattern-triggered immunity (PTI) response. We used an A. thaliana mesophyll protoplast-based assay to identify Xcc8004 T3S effectors that effectively interfere with PTI signalling induced by the bacterial peptide flg22. 11 of the 24 tested effector proteins (XopK, XopQ, HrpW, XopN, XopAC, XopD, XopZ1, XopAG, AvrBs2, XopL and XopX-1) inhibited expression of the flg22-inducible gene FRK1, and five effectors (XopK, XopG, XopQ, XopL and XopX-1) inhibited the expression of the flg22-inducible gene WRKY33. Therefore, there are 12 effector proteins that can inhibit the expression of relevant flg22-inducible genes. It was further investigated whether the 12 effector proteins affect the phosphorylation activation of mitogen-activated protein (MAP) kinases MPK3/MPK6, and four effector proteins (XopK, XopQ, XopZ1 and XopX-1) were found to markedly inhibit MPK3/MPK6 activation. Moreover, a subcellular localisation analysis revealed that the tested effectors were localised within various subcellular compartments. These results indicate that multiple T3S effectors in the Xcc8004 genome interfere with flg22-induced PTI signalling via various molecular mechanisms.

HbWRKY40 plays an important role in the regulation of pathogen resistance in Hevea brasiliensis.

KEY MESSAGE: Overexpression of HbWRKY40 induces ROS burst in tobacco and increases disease resistance in Arabidopsis; RNA-seq and ChIP assays revealed the regulatory network of HbWRKY40 in plant defense. WRKY, a family of plant transcription factors, are involved in the regulation of numerous biological processes. In rubber tree Hevea brasiliensis, the roles of WRKYs remain poorly understood. In the present study, a total of 111 genes encoding putative HbWRKY proteins were identified in the H. brasiliensis genome. Among these genes, HbWRKY40 transcripts were significantly induced by Colletotrichum gloeosporioides and salicylic acid. To assess its roles in plant defense, HbWRKY40 was over-expressed in Nicotiana benthamiana and Arabidopsis thaliana. The results showed that HbWRKY40 significantly induced reactive oxygen species burst in N. benthamiana and increased resistance of Arabidopsis against Botrytis cinerea. Transient expression in mesophyll cell protoplasts of H. brasiliensis showed that HbWRKY40 localizes at nuclei. In addition, transcripts of 145 genes were significantly up-regulated and 6 genes were down-regulated in the protoplasts over-expressing HbWRKY40 based on the RNA-seq analysis. Among these potential downstream targets, 12 genes contain potential WRKY-binding sites at the promoter regions. Further analysis through chromatin immunoprecipitation revealed that 10 of these 12 genes were the downstream targets of HbWRKY40. Taken together, our findings indicate that HbWRKY40 plays an important role in the disease resistance by regulating defense-associated genes in H. brasiliensis.

Predicting Virulence of Fusarium Oxysporum f. sp. Cubense Based on the Production of Mycotoxin Using a Linear Regression Model.

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is one of the most destructive diseases for banana. For their risk assessment and hazard characterization, it is vital to quickly determine the virulence of Foc isolates. However, this usually takes weeks or months using banana plant assays, which demands a better approach to speed up the process with reliable results. Foc produces various mycotoxins, such as fusaric acid (FSA), beauvericin (BEA), and enniatins (ENs) to facilitate their infection. In this study, we developed a linear regression model to predict Foc virulence using the production levels of the three mycotoxins. We collected data of 40 Foc isolates from 20 vegetative compatibility groups (VCGs), including their mycotoxin profiles (LC-MS) and their plant disease index (PDI) values on Pisang Awak plantlets in greenhouse. A linear regression model was trained from the collected data using FSA, BEA and ENs as predictor variables and PDI values as the response variable. Linearity test statistics showed this model meets all linearity assumptions. We used all data to predict PDI with high fitness of the model (coefficient of determination (R2 = 0.906) and adjust coefficient (R2adj = 0.898)) indicating a strong predictive power of the model. In summary, we developed a linear regression model useful for the prediction of Foc virulence on banana plants from the quantification of mycotoxins in Foc strains, which will facilitate quick determination of virulence in newly isolated Foc emerging Fusarium wilt of banana epidemics threatening banana plantations worldwide.

Photosynthesis is sensitive to nitric oxide and respiration sensitive to hydrogen peroxide: Studies with pea mesophyll protoplasts.

Reports on the effect of nitric oxide (NO) or reactive oxygen species (ROS) on photosynthesis and respiration in leaf tissues are intriguing; therefore, the effects of exogenous addition of sodium nitroprusside (SNP, releases NO) or H2O2 on the photosynthetic O2 evolution and respiratory O2 uptake by mesophyll protoplasts in pea (Pisum sativum) were evaluated in the present study. Low concentrations of SNP or H2O2 were used to minimize nonspecific effects. The effects of NO or H2O2 on respiration and photosynthesis were different. The presence of NO decreased the rate of photosynthesis but caused a marginal stimulation of dark respiration. Conversely, externally administered H2O2 drastically decreased the rate of respiration but only slightly decreased photosynthesis. The PS I activity was more sensitive to NO than PS II. On the other hand, 100 µM H2O2 had no effect on the photochemical reactions of either PS I or PS II. The sensitivity of photosynthesis to antimycin A or SHAM (reflecting the interplay between chloroplasts and mitochondria) was not affected by NO. By contrast, H2O2 markedly decreased the sensitivity of photosynthesis to antimycin A and SHAM. It can be concluded that chloroplasts are the primary targets of NO, while mitochondria are the primary targets of ROS in plant cells. We propose that H2O2 can be an important signal to modulate the crosstalk between chloroplasts and mitochondria.

Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4.

Fusaric acid (FSA) is a phytotoxin produced by several Fusarium species and has been associated with plant disease development, although its role is still not well understood. Mutation of key genes in the FSA biosynthetic gene (FUB) cluster in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) reduced the FSA production, and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants. When pretreated with FSA, both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion. Banana embryogenic cell suspensions (ECSs) treated with FSA exhibited a lower rate of O2 uptake, loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) accumulation, and greater nuclear condensation and cell death. Consistently, transcriptomic analysis of FSA-treated ECSs showed that FSA may induce plant cell death through regulating the expression of genes involved in mitochondrial functions. The results herein demonstrated that the FSA from Foc TR4 functions as a positive virulence factor and acts at the early stage of the disease development before the appearance of the fungal hyphae in the infected tissues.

PatJAZ6 Acts as a Repressor Regulating JA-Induced Biosynthesis of Patchouli Alcohol in Pogostemon Cablin.

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.

Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.

Identification of Potential Auxin-Responsive Small Signaling Peptides through a Peptidomics Approach in Arabidopsis thaliana.

Small signaling peptides (SSPs) are a class of short peptides playing critical roles in plant growth and development. SSPs are also involved in the phytohormone signaling pathway. However, identification of mature SSPs is still a technical challenge because of their extremely low concentrations in plant tissue and complicated interference by many other metabolites. Here, we report an optimized protocol to extract SSPs based on protoplast extraction and to analyze SSPs based on tandem mass spectrometry peptidomics. Using plant protoplasts as the material, soluble peptides were directly extracted into phosphate buffer. The interference of non-signaling peptides was significantly decreased. Moreover, we applied the protocol to identify potential SSPs in auxin treated wild type and auxin biosynthesis defective mutant yuc2yuc6. Over 100 potential SSPs showed a response to auxin in Arabidopsis thaliana.

A Novel Biological Agent Cytosinpeptidemycin Inhibited the Pathogenesis of Tobacco Mosaic Virus by Inducing Host Resistance and Stress Response.

Cytosinpeptidemycin (CytPM) is a microbial pesticide that displayed broad-spectrum antiviral activity against various plant viruses. However, the molecular mechanism underlying antiviral activity of CytPM is poorly understood. In this study, the results demonstrated that CytPM could effectively delay the systemic infection of tobacco mosaic virus (TMV) in Nicotiana benthamiana and significantly inhibit the viral accumulation in tobacco BY-2 protoplasts. Results of RNA-seq indicated that 210 and 120 differential expressed genes (DEGs) were significantly up- and down-regulated after CytPM treatment in BY-2 protoplasts, respectively. In addition, KEGG analysis indicated that various DEGs were involved in endoplasmic reticulum (ER) protein processing, suggesting a possible correlation between ER homeostasis and virus resistance. RT-qPCR was performed to validate the gene expression of crucial DEGs related with defense, stress responses, signaling transduction, and phytohormone, which were consistent with results of RNA-seq. Our works provided valuable insights into the antiviral mechanism of CytPM that induced host resistance to viral infection.

Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH, Ca2+ and GABA.

We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.