A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Light quality-dependent roles of REVEILLE proteins in the circadian system.

Several closely related Myb-like activator proteins are known to have partially redundant functions within the plant circadian clock, but their specific roles are not well understood. To clarify the function of the REVEILLE 4, REVEILLE 6, and REVEILLE 8 transcriptional activators, we characterized the growth and clock phenotypes of CRISPR-Cas9-generated single, double, and triple rve mutants. We found that these genes act synergistically to regulate flowering time, redundantly to regulate leaf growth, and antagonistically to regulate hypocotyl elongation. We previously reported that increasing intensities of monochromatic blue and red light have opposite effects on the period of triple rve468 mutants. Here, we further examined light quality-specific phenotypes of rve mutants and report that rve468 mutants lack the blue light-specific increase in expression of some circadian clock genes observed in wild type. To investigate the basis of these blue light-specific circadian phenotypes, we examined RVE protein abundances and degradation rates in blue and red light and found no significant differences between these conditions. We next examined genetic interactions between RVE genes and ZEITLUPE and ELONGATED HYPOCOTYL5, two factors with blue light-specific functions in the clock. We found that the RVEs interact additively with both ZEITLUPE and ELONGATED HYPOCOTYL5 to regulate circadian period, which suggests that neither of these factors are required for the blue light-specific differences that we observed. Overall, our results suggest that the RVEs have separable functions in plant growth and circadian regulation and that they are involved in blue light-specific circadian signaling via a novel mechanism.

Rastonia solanacearum type â ¢ effectors target host 14-3-3 proteins to suppress plant immunity.

14-3-3 proteins play important roles in plant metabolism and stress response. Tomato 14-3-3 proteins, SlTFT4 and SlTFT7, serve as hubs of plant immunity and are targeted by some pathogen effectors. Ralstonia solanacearum with more than 70 type Ⅲ effectors (T3Es) is one of the most destructive plant pathogens. However, little is known on whether R. solanacearum T3Es target SlTFT4 and SlTFT7 and hence interfere with plant immunity. We first detected the associations of SlTFT4/SlTFT7 with R. solanacearum T3Es by luciferase complementation assay, and then confirmed the interactions by yeast two-hybrid approach. We demonstrated that 22 Ralstonia T3Es were associated with both SlTFT4 and SlTFT7, and five among them suppressed the hypersensitive response induced by MAPKKKα, a protein kinase which associated with SlTFT4/SlTFT7. We further demonstrated that suppression of MAPKKKα-induced HR and plant basal defense by the T3E RipAC depend on its association with 14-3-3 proteins. Our findings firstly demonstrate that R. solanacearum T3Es can manipulate plant immunity by targeting 14-3-3 proteins, SlTFT4 and SlTFT7, providing new insights into plant-R. solanacearum interactions.

ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves.

5-Aminolevulinic acid (ALA), known as a new natural plant growth regulator, can reverse abscisic acid (ABA)-induced stomatal closure. The protein phosphatase 2A (PP2A) played an important role in regulation of stomatal movement by ALA and ABA; however, the underlying molecular mechanisms remain unclear. Here, we report that ALA promotes MdPP2A activity and gene expression in the leaf epidermis of apple (Malus × domestica Borkh.), and expression of the catalytic subunit MdPP2AC was most significantly correlated with stomatal aperture. Western blotting showed that ALA enhanced MdPP2AC protein abundance and phosphorylation. Y2H (yeast two hybrid), FLC (firefly luciferase complementation imaging) and BiFC (Bimolecular fluorescence complementation) assays showed that MdPP2AC interacted with several other MdPP2A subunits as well as MdSnRK2.6 (Sucrose non-fermenting 1-related protein kinase 2.6), and the latter interaction was further verified by pull-down and MST (microscale thermophoresis) assays. ALA downregulated ABA-induced MdSnRK2.6 gene expression, kinase activity, and protein phosphorylation. In transiently transgenic apple leaves, OE-MdPP2AC promoted stomatal aperture by reducing Ca2+ and H2O2 levels but increasing flavonol levels in guard cells. Conversely, OE-MdSnRK2.6 induced stomatal closure by increasing Ca2+ and H2O2 but reducing flavonols. Partial silencing of these genes had opposite effects on Ca2+, H2O2, flavonols, and stomatal movement. Application of exogenous ALA stimulated PP2A activity, which promoted SnRK2.6 dephosphorylation and lower kinase activity in wild-type and transgenic apple leaves. We therefore propose that PP2AC, which dephosphorylates SnRK2.6 and represses its enzyme activity, mediates ALA signaling to inhibit ABA-induced stomatal closure in apple leaves.

MYB44 regulates PTI by promoting the expression of EIN2 and MPK3/6 in Arabidopsis.

The plant signaling pathway that regulates pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) involves mitogen-activated protein kinase (MAPK) cascades that comprise sequential activation of several protein kinases and the ensuing phosphorylation of MAPKs, which activate transcription factors (TFs) to promote downstream defense responses. To identify plant TFs that regulate MAPKs, we investigated TF-defective mutants of Arabidopsis thaliana and identified MYB44 as an essential constituent of the PTI pathway. MYB44 confers resistance against the bacterial pathogen Pseudomonas syringae by cooperating with MPK3 and MPK6. Under PAMP treatment, MYB44 binds to the promoters of MPK3 and MPK6 to activate their expression, leading to phosphorylation of MPK3 and MPK6 proteins. In turn, phosphorylated MPK3 and MPK6 phosphorylate MYB44 in a functionally redundant manner, thus enabling MYB44 to activate MPK3 and MPK6 expression and further activate downstream defense responses. Activation of defense responses has also been attributed to activation of EIN2 transcription by MYB44, which has previously been shown to affect PAMP recognition and PTI development. AtMYB44 thus functions as an integral component of the PTI pathway by connecting transcriptional and posttranscriptional regulation of the MPK3/6 cascade.

The Ralstonia solanacearum Type III Effector RipAW Targets the Immune Receptor Complex to Suppress PAMP-Triggered Immunity.

Bacterial wilt, caused by Ralstonia solanacearum, one of the most destructive phytopathogens, leads to significant annual crop yield losses. Type III effectors (T3Es) mainly contribute to the virulence of R. solanacearum, usually by targeting immune-related proteins. Here, we clarified the effect of a novel E3 ubiquitin ligase (NEL) T3E, RipAW, from R. solanacearum on pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and further explored its action mechanism. In the susceptible host Arabidopsis thaliana, we monitored the expression of PTI marker genes, flg22-induced ROS burst, and callose deposition in RipAW- and RipAWC177A-transgenic plants. Our results demonstrated that RipAW suppressed host PTI in an NEL-dependent manner. By Split-Luciferase Complementation, Bimolecular Fluorescent Complimentary, and Co-Immunoprecipitation assays, we further showed that RipAW associated with three crucial components of the immune receptor complex, namely FLS2, XLG2, and BIK1. Furthermore, RipAW elevated the ubiquitination levels of FLS2, XLG2, and BIK1, accelerating their degradation via the 26S proteasome pathway. Additionally, co-expression of FLS2, XLG2, or BIK1 with RipAW partially but significantly restored the RipAW-suppressed ROS burst, confirming the involvement of the immune receptor complex in RipAW-regulated PTI. Overall, our results indicate that RipAW impairs host PTI by disrupting the immune receptor complex. Our findings provide new insights into the virulence mechanism of R. solanacearum.

COP1 Mediates Dark-Induced Stomatal Closure by Suppressing FT, TSF and SOC1 Expression to Promote NO Accumulation in Arabidopsis Guard Cells.

RING-finger-type ubiquitin E3 ligase Constitutively Photomorphogenic 1 (COP1) and floral integrators such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) have been identified as regulators of stomatal movement. However, little is known about their roles and relationship in dark-induced stomatal closure. Here, we demonstrated that COP1 is required for dark-induced stomatal closure using cop1 mutant. The cop1 mutant closed stomata in response to exogenous nitric oxide (NO) but not hydrogen peroxide (H2O2), and H2O2 but not NO accumulated in cop1 in darkness, further indicating that COP1 acts downstream of H2O2 and upstream of NO in dark-induced stomatal closure. Expression of FT, TSF and SOC1 in wild-type (WT) plants decreased significantly with dark duration time, but this process was blocked in cop1. Furthermore, ft, tsf, and soc1 mutants accumulated NO and closed stomata faster than WT plants in response to darkness. Altogether, our results indicate that COP1 transduces H2O2 signaling, promotes NO accumulation in guard cells by suppressing FT, TSF and SOC1 expression, and consequently leads to stomatal closure in darkness. These findings add new insights into the mechanisms of dark-induced stomatal closure.

Phosphorylation of a wheat aquaporin at two sites enhances both plant growth and defense.

Eukaryotic aquaporins share the characteristic of functional multiplicity in transporting distinct substrates and regulating various processes, but the underlying molecular basis for this is largely unknown. Here, we report that the wheat (Triticum aestivum) aquaporin TaPIP2;10 undergoes phosphorylation to promote photosynthesis and productivity and to confer innate immunity against pathogens and a generalist aphid pest. In response to elevated atmospheric CO2 concentrations, TaPIP2;10 is phosphorylated at the serine residue S280 and thereafter transports CO2 into wheat cells, resulting in enhanced photosynthesis and increased grain yield. In response to apoplastic H2O2 induced by pathogen or insect attacks, TaPIP2;10 is phosphorylated at S121 and this phosphorylated form transports H2O2 into the cytoplasm, where H2O2 intensifies host defenses, restricting further attacks. Wheat resistance and grain yield could be simultaneously increased by TaPIP2;10 overexpression or by expressing a TaPIP2;10 phosphomimic with aspartic acid substitutions at S121 and S280, thereby improving both crop productivity and immunity.

Three amino acid residues are required for the recognition of Ralstonia solanacearum RipTPS in Nicotiana tabacum.

Ralstonia solanacearum causes devastating diseases in a wide range of economically important crops. It secretes a large number of virulence factors, also known as effectors, to promote its infection, and some of them are recognized when the host plant contains corresponding resistance genes. In this study we showed that a type III effector RipTPS from the avirulent R. solanacearum strain GMI1000 (RipTPSG) specifically induced cell death in Nicotiana tabacum, but not in Nicotiana benthamiana, whereas the RipTPS homolog in the virulent strain CQPS-1 (RipTPSC) induced cell death in neither N. tabacum nor N. benthamiana. These results indicated that RipTPSG is recognized in N. tabacum. Expression of RipTPSG induced upregulation of hypersensitive response (HR) -related genes in N. tabacum. The virulence of CQPS-1 was reduced when RipTPSG was genetically introduced into CQPS-1, further confirming that RipTPSG functions as an avirulence determinant. Protein sequence alignment indicated that there are only three amino acid polymorphisms between RipTPSG and RipTPSC. Site-directed mutagenesis analyses confirmed that the three amino acid residues are jointly required for the recognition of RipTPSG in N. tabacum. Expression of either RipTPSG or RipTPSC suppressed flg22-triggered reactive oxygen species (ROS) burst in N. benthamiana, suggesting that RipTPS contributes to pathogen virulence. Mutating the conserved residues in RipTPS's trehalose-phosphate synthase (TPS) domain did not block its HR induction and defense suppression activity, indicating that the TPS activity is not required for RipTPS's avirulence and virulence function.

A conserved type III effector RipB is recognized in tobacco and contributes to Ralstonia solanacearum virulence in susceptible host plants.

Ralstonia solanacearum, the causal agent of bacterial wilt, causes devastating diseases in a wide range of plants including potato, tomato, pepper and tobacco. The pathogen delivers approximately 70 type III effectors (T3Es) into plant cells during infection. In this study, we confirmed that a T3E RipB is recognized in tobacco. We further demonstrated that RipB is conserved among R. solanacearum isolates and five different ripB alleles are all recognized in tobacco. The ripB from GMI1000 was transformed into susceptible host Arabidopsis, and a defect in root development was observed in ripB-transgenic plants. Pathogen inoculation assays showed that ripB expression promoted plant susceptibility to R. solanacearum infection, indicating that RipB contributes to pathogen virulence in Arabidopsis. Expression of ripB in roq1 mutant partially suppressed reactive oxygen species production, confirming that RipB interferes with plant basal defense. Interestingly, ripB expression promoted cytokinin-related gene expression in Arabidopsis, suggesting a role of cytokinin signaling pathway in plant-R. solanacearum interactions. Finally, RipB harbors potential 14-3-3 binding motifs, but the associations between RipB and 14-3-3 proteins were undetectable in yeast two-hybrid assay. Together, our results demonstrate that multiple ripB alleles are recognized in Nicotiana, and RipB suppresses basal defense in susceptible host to promote R. solanacearum infection.

Ethylene Acts as a Local and Systemic Signal to Mediate UV-B-Induced Nitrate Reallocation to Arabidopsis Leaves and Roots via Regulating the ERFs-NRT1.8 Signaling Module.

Nitrate is the preferred nitrogen source for plants and plays an important role in plant growth and development. Under various soil stresses, plants reallocate nitrate to roots to promote stress tolerance through the ethylene-ethylene response factors (ERFs)-nitrate transporter (NRT) signaling module. As a light signal, ultraviolet B (UV-B) also stimulates the production of ethylene. However, whether UV-B regulates nitrate reallocation in plants via ethylene remains unknown. Here, we found that UV-B-induced expression of ERF1B, ORA59, ERF104, and NRT1.8 in both Arabidopsis shoots and roots as well as nitrate reallocation from hypocotyls to leaves and roots were impaired in ethylene signaling mutants for Ethylene Insensitive2 (EIN2) and EIN3. UV-B-induced NRT1.8 expression and nitrate reallocation to leaves and roots were also inhibited in the triple mutants for ERF1B, ORA59, and ERF104. Deletion of NRT1.8 impaired UV-B-induced nitrate reallocation to both leaves and roots. Furthermore, UV-B promoted ethylene release in both shoots and roots by enhancing the gene expression and enzymatic activities of ethylene biosynthetic enzymes only in shoots. These results show that ethylene acts as a local and systemic signal to mediate UV-B-induced nitrate reallocation from Arabidopsis hypocotyls to both leaves and roots via regulating the gene expression of the ERFs-NRT1.8 signaling module.

MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples.

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the 'Fuji' apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.

A BTB/POZ domain-containing protein negatively regulates plant immunity in Nicotiana benthamiana.

Plants have evolved immune systems to fight against pathogens. However, it is still largely unknown how the plant immunity is finely regulated. Here we identified a BTB/POZ domain-containing protein, namely NbBTB, which is predicted to be a member of the ubiquitin E3 ligase complex. The NbBTB expression is downregulated upon the oomycete pathogen Phytophthora parasitica infection. Overexpression of NbBTB in Nicotiana benthamiana promoted plant susceptibility to P. parasitica infection, and silencing NbBTB increased plant resistance to P. parasitica, indicating that NbBTB negatively modulates plant basal defense. Interestingly, overexpressing or silencing NbBTB did not affect plant resistance to two bacterial pathogens Ralstonia solanacearum and Pseudomonas syringae, suggesting that NbBTB is specifically involved in basal defense against oomycete pathogen. Expression of NbBTB suppressed hypersensitive response (HR) triggered by avirulence proteins from both R. sonanacearum and P. infestans, and silencing NbBTB showed the opposite effect, indicating that NbBTB negatively regulates effector-triggered immunity (ETI). Protein accumulation of avirulence effectors in NbBTB-silenced plants was significantly enhanced, suggesting that NbBTB is likely to negatively modulate ETI by affecting effector protein accumulation. Together, our results demonstrated that NbBTB is a negative regulator in both plant basal defense and ETI.

Transcriptomic Profiling of Apple Calli With a Focus on the Key Genes for ALA-Induced Anthocyanin Accumulation.

The red color is an attractive trait of fruit and determines its market acceptance. 5-Aminolevulinic acid (ALA), an eco-friendly plant growth regulator, has played a universal role in plant secondary metabolism regulation, particularly in flavonoid biosynthesis. It has been widely reported that ALA can up-regulate expression levels of several structural genes related to flavonoid metabolism and anthocyanin accumulation. However, the molecular mechanisms behind ALA-induced expression of these genes are complicated and still far from being completely understood. In this study, transcriptome analysis identified the differentially expressed genes (DEGs) associated with ALA-induced anthocyanin accumulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the flavonoid biosynthesis (ko00941) pathway was significantly enhanced in the ALA-treated apple calli at 24, 48, and 72 h after the treatment. Expression pattern revealed that ALA up-regulated the expression of the structural genes related to not only anthocyanin biosynthesis (MdCHS, MdCHI, MdF3'H, MdDFR, MdANS, and MdUFGT) but also anthocyanin transport (MdGST and MdMATE). Two R2R3-MYB transcription factors (MdMYB10 and MdMYB9), which are the known positive regulators of anthocyanin biosynthesis, were significantly induced by ALA. Gene overexpression and RNA interference assays demonstrated that MdMYB10 and MdMYB9 were involved in ALA-induced anthocyanin biosynthesis. Moreover, MdMYB10 and MdMYB9 might positively regulate the transcription of MdMATE8 by binding to the promoter region. These results indicate that MdMYB10 and MdMYB9 modulated structural gene expression of anthocyanin biosynthesis and transport in response to ALA-mediated apple calli coloration at the transcript level. We herein provide new details regarding transcriptional regulation of ALA-induced color development.

Photosynthetic Responses of Canola to Exogenous Application or Endogenous Overproduction of 5-Aminolevulinic Acid (ALA) under Various Nitrogen Levels.

Limited data are available on the effects of 5-aminolevulinic acid (ALA) on plant photosynthesis in relation to the nitrogen (N) level. In this study, we investigate photosynthetic responses to ALA in canola plants (Brassica napus L.). We used wild-type plants without ALA addition (controls), wild-type plants with exogenous ALA application, and transgenic plants that endogenously overproduced ALA. The plants were grown hydroponically in nutrient solutions with low, middle, and high concentrations of N. Our results indicate that plants in both treatment groups had higher chlorophyll contents and net photosynthetic rates and lower intracellular CO2 concentrations in the leaves, as compared to controls. Furthermore, simultaneous measurement of prompt chlorophyll fluorescence and modulated 820-nm reflections showed that the active photosystem II (PS II) reaction centers, electron transfer capacity, and photosystem I (PS I) activity were all higher in treated plants than controls at all N levels; however, the responses of some photochemical processes to ALA were significantly affected by the N level. For example, under low N conditions only, a negative ΔK peak appeared in the prompt chlorophyll fluorescence curve, indicating a protective effect of ALA on electron donation via activation of the oxygen-evolving complex. Taken together, our findings suggest that ALA contributes to the promotion of photosynthesis by regulating photosynthetic electron transport under various N levels. These findings may provide a new strategy for improving photosynthesis in crops grown in N-poor conditions or reduced N-fertilization requirements.

Genome-wide identification and comparative analysis of GST gene family in apple (Malus domestica) and their expressions under ALA treatment.

Anthocyanins, a subclass of flavonoids, are synthesized at the cytoplasmic surface of the endoplasmic reticulum (ER), which then accumulate in vacuoles. Plant glutathione S-transferase (GST) genes are involved in anthocyanin transportation. Here, a total of 52, 42, 50, and 29 GST genes were identified from apple, pear, peach, and strawberry, respectively, through a comprehensive genome-wide survey. Based on phylogenetic analyses, the GST proteins of the four crops could be divided into the classes Phi, Tau, DHAR, TCHQD, and Lambda. The structure and chromosomal distribution of apple GST genes were further analyzed. The GST gene family expansion in apple likely occurred through tandem duplications, and purifying selection played a pivotal role in the evolution of GST genes. Synteny analysis showed strong microsynteny between apple and Arabidopsis/strawberry, but no microsynteny was detected between apple/strawberry/Arabidopsis and rice. Aminolevulinic acid (ALA), a key precursor of tetrapyrrole compounds, can significantly improve anthocyanin accumulation in fruits, Using RNA-seq and qRT-PCR analysis, we found that ALA treatment led to the differential expression of GST genes in apples. MdGSTF12 was strongly induced by ALA, suggesting that MdGSTF12 may play a role in ALA-induced anthocyanin accumulation. These results provide a detailed overview of GST genes in four Rosaceae species and indicate that GSTs are involved in ALA-induced anthocyanin accumulation.

Transcriptomic Analysis of Ficus carica Peels with a Focus on the Key Genes for Anthocyanin Biosynthesis.

Fig (Ficus carica L.), a deciduous fruit tree of the Moraceae, provides ingredients for human health such as anthocyanins. However, little information is available on its molecular structure. In this study, the fig peels in the yellow (Y) and red (R) stages were used for transcriptomic analyses. Comparing the R with the Y stage, we obtained 6224 differentially expressed genes, specifically, anthocyanin-related genes including five CHS, three CHI, three DFR, three ANS, two UFGT and seven R2R3-MYB genes. Furthermore, three anthocyanin biosynthetic genes, i.e., FcCHS1, FcCHI1 and FcDFR1, and two R2R3-MYB genes, i.e., FcMYB21 and FcMYB123, were cloned; sequences analysis and their molecular characteristics indicated their important roles in fig anthocyanin biosynthesis. Heterologous expression of FcMYB21 and FcMYB123 significantly promoted anthocyanin accumulation in both apple fruits and calli, further suggesting their regulatory roles in fig coloration. These findings provide novel insights into the molecular mechanisms behind fig anthocyanin biosynthesis and coloration, facilitating the genetic improvement of high-anthocyanin cultivars and other horticultural traits in fig fruits.

Exogenous 5-aminolevulinic acid improves strawberry tolerance to osmotic stress and its possible mechanisms.

Cultivated strawberry, one of the major fruit crops worldwide, is an evergreen plant with shallow root system, and thus sensitive to environmental changes, including drought stress. To investigate the effect of 5-aminolevulinic acid (ALA), a new environment-friendly plant growth regulator, on strawberry drought tolerance and its possible mechanisms, we treated strawberry (Fragaria × annanasa Duch. cv. 'Benihoppe') with 15% polyethylene glycol 6000 to simulate osmotic stress with or without 10 mg l-1 ALA. We found that ALA significantly alleviated PEG-inhibited plant growth and improved water absorption and xylem sap flux, indicating ALA mitigates the adverse effect of osmotic stress on strawberry plants. Gas exchange and chlorophyll fluorescence analysis showed that ALA mitigated PEG-induced decreases of Pn , Gs , Tr , Pn /Ci , photosystem I and II reaction center activities, electron transport activity, and photosynthetic performance indexes. Equally important, ALA promoted PEG-increased antioxidant enzyme activities and repressed PEG-increased malondialdehyde and superoxide anion in both leaves and roots. Specially, ALA repressed H2 O2 increase in leaves, but stimulated it in roots. Furthermore, ALA repressed abscisic acid (ABA) biosynthesis and signaling gene expressions in leaves, but promoted those in roots. In addition, ALA blocked PEG-downregulated expressions of plasmalemma and tonoplast aquaporin genes PIP and TIP in both leaves and roots. Taken together, ALA effectively enhances strawberry drought tolerance and the mechanism is related to the improvement of water absorption and conductivity. The tissue-specific responses of ABA biosynthesis, ABA signaling, and H2 O2 accumulation to ALA in leaves and roots play key roles in ALA-improved strawberry tolerance to osmotic stress.

PP2A and microtubules function in 5-aminolevulinic acid-mediated H2 O2 signaling in Arabidopsis guard cells.

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H2 O2 . However, the mechanisms behind ALA-decreased H2 O2 in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H2 O2 in guard cell signaling. Finally, we demonstrate the role of H2 O2 -mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H2 O2 decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.

Hydrogen peroxide as a mediator of 5-aminolevulinic acid-induced Na+ retention in roots for improving salt tolerance of strawberries.

To explore the mechanisms of 5-aminolevulinic acid (ALA)-improved plant salt tolerance, strawberries (Fragaria × ananassa Duch. cv. 'Benihoppe') were treated with 10 mg l-1 ALA under 100 mmol l-1 NaCl stress. We found that the amount of Na+ increased in the roots but decreased in the leaves. Laser scanning confocal microscopy (LSCM) observations showed that ALA-induced roots had more Na+ accumulation than NaCl alone. Measurement of the xylem sap revealed that ALA repressed Na+ concentrations to a large extent. The electron microprobe X-ray assay also confirmed ALA-induced Na+ retention in roots. qRT-PCR showed that ALA upregulated the gene expressions of SOS1 (encoding a plasma membrane Na+ /H+ antiporter), NHX1 (encoding a vacuolar Na+ /H+ antiporter) and HKT1 (encoding a protein of high-affinity K+ uptake), which are associated with Na+ exclusion in the roots, Na+ sequestration in vacuoles and Na+ unloading from the xylem vessels to the parenchyma cells, respectively. Furthermore, we found that ALA treatment reduced the H2 O2 content in the leaves but increased it in the roots. The exogenous H2 O2 promoted plant growth, increased root Na+ retention and stimulated the gene expressions of NHX1, SOS1 and HKT1. Diphenyleneiodonium (DPI), an inhibitor of H2 O2 generation, suppressed the effects of ALA or H2 O2 on Na+ retention, gene expressions and salt tolerance. Therefore, we propose that ALA induces H2 O2 accumulation in roots, which mediates Na+ transporter gene expression and more Na+ retention in roots, thereby improving plant salt tolerance.