Tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of the PpPGF to regulate peach softening during fruit ripening.

Peach fruit rapidly soften after harvest, a significant challenge for producers and marketers as it results in rotting fruit and significantly reduces shelf life. In this study, we identified two tandem genes, PpNAC1 and PpNAC5, within the sr (slow ripening) locus. Phylogenetic analysis showed that NAC1 and NAC5 are highly conserved in dicots and that PpNAC1 is the orthologous gene of Non-ripening (NOR) in tomato. PpNAC1 and PpNAC5 were highly expressed in peach fruit, with their transcript levels up-regulated at the onset of ripening. Yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC1 interacting with PpNAC5 and this interaction occurs with the tomato and apple orthologues. Transient gene silencing experiments showed that PpNAC1 and PpNAC5 positively regulate peach fruit softening. Yeast one-hybrid and dual luciferase assays and LUC bioluminescence imaging proved that PpNAC1 and PpNAC5 directly bind to the PpPGF promoter and activate its transcription. Co-expression of PpNAC1 and PpNAC5 showed higher levels of PpPGF activation than expression of PpNAC1 or PpNAC5 alone. In summary, our findings demonstrate that the tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of PpPGF to regulate fruit softening during peach fruit ripening.

A rapid and efficient Agrobacterium-mediated transient transformation system in grape berries.

Transient transformation is extremely useful for rapid in vivo assessment of gene function, especially for fruit-related genes. Grape berry, while an important fruit crop, is recalcitrant to transient transformation, due to the high turgor pressure in its mesocarp cells that limits the ability of Agrobacterium to penetrate into the tissue. It is urgent to establish a simple transient transformation system for rapid analysis of gene function. In this study, different injection methods, grape genotypes, and developmental stages were tested in order to develop a rapid and efficient Agrobacterium-mediated transient transformation methodology for grape berries. Two injection methods, namely punch injection and direct injection, were evaluated using the ß-glucuronidase (GUS) gene and by x-gluc tissue staining and 4-methylumbelliferyl-ß-D-glucuronide fluorescence analysis. The results indicated that there were no significant differences on transformation effects between the two methods, but the latter was more suitable because of its simplicity and convenience. Six grape cultivars ('Hanxiangmi', 'Moldova', 'Zijixin', 'Jumeigui', 'Shine-Muscat', and 'A17') were tested for transient transformation. 'Hanxiangmi', 'Moldova', and 'Zijixin' grape berries were not suitable for agroinfiltration due to frequently fruit cracking, browning, and formation of scar skin. The fruit integrity rates of 'Jumeigui', 'Shine-Muscat', and 'A17' berries were all above 80%, and GUS activity was detected in the berries of the three cultivars 3-14 days after injection with the Agrobacterium culture, while higher GUS activities were observed in the 'Jumeigui' berries. The levels of GUS activity in injected berries at 7-8 weeks after full blooming (WAFB) were more than twice at 6 WAFB. In subsequent assays, the over-expression of MYB transcription factor VvMYB44 via transient transformation accelerated the anthocyanin accumulation and fruit coloring through raising the expression levels of VvLAR1, VvUFGT, VvLDOX, VvANS, and VvDFR, which verified the effectiveness of this transformation system. These experiments finally identified the reliable grape cultivars and suitable operational approach for transient transformation and further indicated that this Agrobacterium-mediated transient transformation system was efficient and suitable for the elucidation of gene function in grape berries.

Jujube Witches' Broom Phytoplasma Effector Zaofeng3, a Homologous Effector of SAP54, Induces Abnormal Floral Organ Development and Shoot Proliferation.

Plant-pathogenic phytoplasmas secrete specific virulence proteins into a host plant to modulate plant function for their own benefit. Identification of phytoplasmal effectors is a key step toward clarifying the pathogenic mechanisms of phytoplasma. In this study, Zaofeng3, also known as secreted jujube witches' broom phytoplasma protein 3 (SJP3), was a homologous effector of SAP54 and induced a variety of abnormal phenotypes, such as phyllody, malformed floral organs, witches' broom, and dwarfism in Arabidopsis thaliana. Zaofeng3 can also induce small leaves, dwarfism, and witches' broom in Ziziphus jujuba. Further experiments showed that the three complete α-helix domains predicted in Zaofeng3 were essential for induction of disease symptoms in jujube. Yeast two-hybrid library screening showed that Zaofeng3 mainly interacts with proteins involved in flower morphogenesis and shoot proliferation. Bimolecular fluorescence complementation assays confirmed that Zaofeng3 interacted with these proteins in the whole cell. Overexpression of zaofeng3 in jujube shoot significantly altered the expression patterns of ZjMADS19, ZjMADS47, ZjMADS48, ZjMADS77, and ZjTCP7, suggesting that overexpressing zaofeng3 might induce floral organ malformation and witches' broom by altering the expression of the transcriptional factors involved in jujube morphogenesis.

The apple transcription factor MdZF-HD11 regulates fruit softening by promoting Mdß-GAL18 expression.

Fruit ripening and the associated softening are major determinants of fruit quality and post-harvest shelf life. Although the mechanisms underlying fruit softening have been intensively studied, there are limited reports on the regulation of fruit softening in apples (Malus domestica). Here, we identified a zinc finger homeodomain transcription factor MdZF-HD11that trans-activates the promoter of Mdß-GAL18, which encodes a pectin-degradation enzyme associated with cell wall metabolism. Both MdZF-HD11 and Mdß-GAL18 genes were up-regulated by exogenous ethylene treatment and repressed by 1-methylcyclopropene treatment. Further experiments revealed that MdZF-HD11 binds directly to the Mdß-GAL18 promoter and up-regulates its transcription. Moreover, using transgenic apple fruit calli, we found that overexpression of Mdß-GAL18 or MdZF-HD11 significantly enhanced ß-galactosidase activity, and overexpression of MdZF-HD11 induced the expression of Mdß-GAL18. We also discovered that transient overexpression of Mdß-GAL18 or MdZF-HD11 in 'Golden Delicious' apple significantly increased the release of ethylene, reduced fruit firmness, promoted the transformation of skin color from green to yellow, and accelerated ripening and softening of the fruit. Finally, the overexpression of MdZF-HD11 in tomato also promoted fruit softening. Collectively, these results indicate that ethylene-induced MdZF-HD11 interacts with Mdß-GAL18 to promote the post-harvest softening of apple.

Peach DELLA Protein PpeDGYLA Is Not Degraded in the Presence of Active GA and Causes Dwarfism When Overexpressed in Poplar and Arabidopsis.

Controlling the tree size of fruit species such as peach can reduce the amount of labor and input needed for orchard management. The phytohormone gibberellin (GA) positively regulates tree size by inducing degradation of the GA signaling repressor DELLA. The N-terminal DELLA domain in this protein is critical for its GA-dependent interaction with the GA receptor GID1 and the resulting degradation of the DELLA protein, which allows for growth-promoting GA signaling. In this study, a DELLA family member, PpeDGYLA, contains a DELLA domain but has amino acid changes in three conserved motifs (DELLA into DGYLA, LEQLE into LERLE, and TVHYNP into AVLYNP). In the absence or presence of GA3, the PpeDGYLA protein did not interact with PpeGID1c and was stable in 35S-PpeDGYLA peach transgenic callus. The overexpression of PpeDGYLA in both polar and Arabidopsis showed an extremely dwarfed phenotype, and these transgenic plants were insensitive to GA3 treatment. PpeDGYLA could interact with PpeARF6-1 and -2, supposed growth-promoting factors. It is suggested that the changes in the DELLA domain of PpeDGYLA may, to some extent, account for the severe dwarf phenotype of poplar and Arabidopsis transgenic plants. In addition, our study showed that the DELLA family contained three clades (DELLA-like, DELLA, and DGLLA). PpeDGYLA clustered into the DGLLA clade and was expressed in all of the analyzed tissues. These results lay the foundation for the further study of the repression of tree size by PpeDGYLA.

The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field.

Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.

PpIBH1-1 limits internode elongation of peach shoot in a dose-dependent manner.

Peach [Prunus persica (L.) Batsch] annual shoots grow up quickly, which limits the lighting and ventilation of an orchard. Atypical bHLH proteins IBH1(INCREASED LEAF INCLINATION1 BINDING bHLH1) play substantial roles in regulating cell elongation and plant stature. In this study, three PpIBH1s (PpIBH1-1/-2/-3) were identified in peach genome and contain a conserved AS domain and a characteristic HLH domain. The transcript levels of three PpIBH1s positively correlated with internode length, which gradually increased from apex to base along the peach shoots. This positive correlation was further confirmed in apple and poplar shoots. And the PpIBH1s gene were highly expressed in the shoot tips collected from twelve dwarf peach cultivars (gid1c mutants). In tissue-specific expression analysis, PpIBH1-1 are more highly expressed in tissues at the growth-arrested stage than cell-elongating. Transgenic Arabidopsis lines showed that different plant heights depending on the dose of PpIBH1-1 transcripts. And the dwarfing PpIBH1-1 transgenic lines were caused by the shorted cell length. PpIBH1-1 interacted with two bHLH factors (PpACE2 and PpLP1). These results suggested that PpIBH1-1 probably prevents internode elongation of peach shoots in a dose-dependent manner. Our work provided a foundation for properly controlling the growth of annual peach branches.

PpTCP18 is upregulated by lncRNA5 and controls branch number in peach (Prunus persica) through positive feedback regulation of strigolactone biosynthesis.

Branch number is an important agronomic trait in peach (Prunus persica) trees because plant architecture affects fruit yield and quality. Although breeders can select varieties with different tree architecture, the biological mechanisms underlying architecture remain largely unclear. In this study, a pillar peach ('Zhaoshouhong') and a standard peach ('Okubo') were compared. 'Zhaoshouhong' was found to have significantly fewer secondary branches than 'Okubo'. Treatment with the synthetic strigolactone (SL) GR24 decreased branch number. Transcriptome analysis indicated that PpTCP18 (a homologous gene of Arabidopsis thaliana BRC1) expression was negatively correlated with strigolactone synthesis gene expression, indicating that PpTCP18 may play an important role in peach branching. Yeast one-hybrid, electrophoretic mobility shift, dual-luciferase assays and PpTCP18-knockdown in peach leaf buds indicated that PpTCP18 could increase expression of PpLBO1, PpMAX1, and PpMAX4. Furthermore, transgenic Arabidopsis plants overexpressing PpTCP18 clearly exhibited reduced primary rosette-leaf branches. Moreover, lncRNA sequencing and transient expression analysis revealed that lncRNA5 targeted PpTCP18, significantly increasing PpTCP18 expression. These results provide insights into the mRNA and lncRNA network in the peach SL signaling pathway and indicate that PpTCP18, a transcription factor downstream of SL signaling, is involved in positive feedback regulation of SL biosynthesis. This role of PpTCP18 may represent a novel mechanism in peach branching regulation. Our study improves current understanding of the mechanisms underlying peach branching and provides theoretical support for genetic improvement of peach tree architecture.

The distribution of bioactive gibberellins along peach annual shoots is closely associated with PpGA20ox and PpGA2ox expression profiles.

BACKGROUND: The rapid growth of annual shoots is detrimental to peach production. While gibberellin (GA) promotes the rapid growth of peach shoots, there is limited information on the identity and expression profiles of GA-metabolism genes for this species. RESULTS: All six GA biosynthetic gene families were identified in the peach genome, and the expression profiles of these family members were determined in peach shoots. The upstream biosynthetic gene families have only one or two members (1 CPS, 2 KSs, and 1 KO), while the downstream gene families have multiple members (7 KAOs, 6 GA20oxs, and 5 GA3oxs). Between the two KS genes, PpKS1 showed a relatively high transcript level in shoots, while PpKS2 was undetectable. Among the seven KAO genes, PpKAO2 was highly expressed in shoots, while PpKAO1 and - 6 were weakly expressed. For the six GA20ox genes, both PpGA20ox1 and - 2 were expressed in shoots, but PpGA20ox1 levels were higher than PpGA20ox2. For the five GA3ox genes, only PpGA3ox1 was highly expressed in shoots. Among these biosynthesis genes, PpGA20ox1 and PpGA3ox1 showed a gradual decrease in transcript level along shoots from top to bottom, and a similar trend was observed in bioactive GA1 and GA4 distribution. Among the GA-deactivation genes, PpGA2ox6 was highly expressed in peach shoots. PpGA2ox1 and - 5 transcripts were relatively lower and showed a similar pattern to PpGA20ox1 and PpGA3ox1 in peach shoots. Overexpression of PpGA20ox1, - 2, or PpGA2ox6 in Arabidopsis or tobacco promoted or depressed the plant growth, respectively, while PpGA3ox1 did not affect plant height. Transient expression of PpGA20ox1 in peach leaves significantly increased bioactive GA1 content. CONCLUSIONS: Our results suggest that PpGA20ox and PpGA2ox expression are closely associated with the distribution of active GA1 and GA4 in peach annual shoots. Our research lays a foundation for future studies into ways to effectively repress the rapid growth of peach shoot.

Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance.

The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42 PpHSP20 genes were detected in the peach genome and were randomly distributed onto the eight chromosomes. The primary modes of gene duplication of the PpHSP20s were dispersed gene duplication (DSD) and tandem duplication (TD). PpHSP20s in the same class shared similar motifs. Based on phylogenetic analysis of HSP20s in peach, Arabidopsis thaliana, Glycine max, and Oryza sativa, the PpHSP20s were classified into 11 subclasses, except for two unclassified PpHSP20s. cis-elements related to stress and hormone responses were detected in the promoter regions of most PpHSP20s. Gene expression analysis of 42 PpHSP20 genes revealed that the expression pattern of PpHSP20-32 was highly consistent with shoot length changes in the cultivar 'Zhongyoutao 14', which is a temperature-sensitive semi-dwarf. PpHSP20-32 was selected for further functional analysis. The plant heights of three transgenic Arabidopsis lines overexpressing PpHSP20-32 were significantly higher than WT, although there was no significant difference in the number of nodes. In addition, the seeds of three over-expressing lines of PpHSP20-32 treated with high temperature showed enhanced thermotolerance. These results provide a foundation for the functional characterization of PpHSP20 genes and their potential use in the growth and development of peach.

PpPIF8, a DELLA2-interacting protein, regulates peach shoot elongation possibly through auxin signaling.

Rapid growth of branches in a peach tree restricts the light penetration and air ventilation within the orchard, which lowers fruit quality and promotes the occurrence of diseases and insects. Our previous works showed that PpDELLA1 and PpDELLA2 repress the rapid growth of annual shoots. Proteins that interact with DELLA are vital for its function. In this study, seven PpPIFs (PpPIF1, -2, -3, -4, -6, -7 and -8) were identified in the peach genome and contain a conserved bHLH domain. Among the seven PpPIFs, PpPIF8 interacted with PpDELLA2 through an unknown motif in the C-terminal and/or the bHLH domain. Overexpression of PpPIF8 in Arabidopsis promotes plant height and branch numbers. Hypocotyl elongation was significantly enhanced by PpPIF8 under weak light intensity. PpPIF8 overexpressed in Arabidopsis and transiently expressed in peach seedlings upregulated the transcription of YUCCA and SAUR19 and downregulated SHY1 and -2. Additionally, PpPIF4 and -8 were significantly induced by weak light. Phylogentic analysis and intron patterns of the bHLH domain strongly suggested that PIFs from six species could be divided into two groups of different evolutionary origins. These results lay a foundation for the further study of the repression of shoot growth by PpDELLA2 through protein interaction with PpPIF8 in peach.

Overexpressing VvWRKY18 from grapevine reduces the drought tolerance in Arabidopsis by increasing leaf stomatal density.

The growth of grapevine [Vitis vinifera L.] is commonly limited by drought stress. The mechanisms by which grapevine copes with drought stress have not yet been extensively clarified. In this study, the drought and abscisic acid (ABA)-induced gene VvWRKY18 was demonstrated to decreased drought tolerance of Arabidopsis thaliana overexpression (VvWRKY18-OE) lines. Compared to wild-type plants, VvWRKY18-OE lines showed increased levels of malonaldehyde (MDA) and the reactive oxygen species (ROS) H2O2 and O2- decreased levels of proline, weakened activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and decreased sensitivity to ABA with respect to stomatal closure.VvWRKY18-OE lines also showed an increase in stomatal density and a higher water loss rate. Negative regulators of stomatal development including SDD1, YDA, TMM, and MPK6, were downregulated in VvWRKY18-OE lines. Transcript levels of the stress-related genes DREB1A and CBF2 were significantly reduced in VvWRKY18-OE lines under drought stress. Taken together, these findings demonstrate that VvWRKY18 reduced drought tolerance in Arabidopsis. Our results contribute to understanding of the roles that WRKY genes play in drought stress and stomatal development.

Multiple indeterminate domain (IDD)-DELLA1 complexes participate in gibberellin feedback regulation in peach.

KEY MESSAGE: Peach encodes 14 INDETERMINATE DOMAIN (IDD) transcription factors. PpIDD4, -12 and -13 mediated PpDELLA1 binding to the PpGA20ox1 promoter. Each of these three PpIDD-DELLA1 complexes activated transcription of PpGA20ox1. PpTPR1 and -4 interrupted the interaction of PpIDDs with PpDELLA1. The plant growth regulator gibberellin (GA) plays an important role in the rapid growth of annual shoots in peach. Our previous study showed that the peach cultivar 'FenHuaShouXingTao' (FHSXT), a gibberellic acid receptor (gid1) mutant, accumulates active GAs in annual shoot tips. This mutant enhances GA feedback regulation in peach. The results of this study suggested that the PpIDD-DELLA1 complex is the underlying mechanism of GA feedback regulation in peach. Fourteen IDD genes were identified in peach, and three PpIDDs (PpIDD4, -12 and -13, all from group IV) interacted with PpDELLA1, an important component in GA signaling pathway. Truncation, segmentation and site mutation of the promoter of PpGA20ox1 (a GA biosynthesis gene) showed that all three PpIDD proteins recognized the core motif TTGTC. PpIDD4 and -13 mainly bind to site 3, while PpIDD12 binds to site 5 of the PpGA20ox1 promoter. All three PpIDD-DELLA1 complexes activated the PpGA20ox1 promoter-LUC fusion. These data suggested that PpIDDs bridge PpDELLA1 and the promoter of PpGA20ox1, which then activated the transcription of PpGA20ox1. In addition, PpTPR1 and -4 disrupted the interaction of PpIDDs with PpDELLA1. Our research will be helpful for understanding and possibly modifying the regulation of annual shoot growth and GA biosynthesis.

Phytoplasma effector Zaofeng6 induces shoot proliferation by decreasing the expression of ZjTCP7 in Ziziphus jujuba.

The jujube witches' broom (JWB) phytoplasma is associated with witches' broom, dwarfism, and smaller leaves in jujube, resulting in yield losses. In this study, eight putative JWB effector proteins were identified from potential mobile units of the JWB genome. Among them, Zaofeng6 induced witches' broom symptoms in Arabidopsis and jujube. Zaofeng6-overexpressing Arabidopsis and unrooted jujube transformants displayed witches' broom-like shoot proliferation. Transient expression of Zaofeng6 induced hypersensitive response like cell death and expression of hypersensitive response marker genes, like harpin-induced gene 1 (H1N1), and the pathogenesis-related genes PR1, PR2, and PR3 in transformed Nicotiana benthamiana leaves, suggesting that Zaofeng6 could be a virulence effector. Yeast two-hybrid library screening and bimolecular fluorescence complementation confirmed that Zaofeng6 interacts with ZjTCP7 through its first two α-helix domains in the cell nuclei. ZjTCP7 mRNA and protein abundance decreased in Zaofeng6 transgenic jujube seedlings. The expression of some genes in the strigolactone signaling pathway (ZjCCD7, ZjCCD8, and CYP711A1) were down-regulated in jujube shoots overexpressing Zaofeng6 and in zjtcp7 CRISPR/Cas9 mutants. Zaofeng6 induces shoot proliferation through decreased expression of ZjTCP7 at the transcriptional and translational levels.

A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system.

Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed species-specific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.

De novo chromosome-level genome of a semi-dwarf cultivar of Prunus persica identifies the aquaporin PpTIP2 as responsible for temperature-sensitive semi-dwarf trait and PpB3-1 for flower type and size.

Peach (Prunus persica) is one of the most important fruit crops globally, but its cultivation can be hindered by large tree size. 'Zhongyoutao 14' (CN14) is a temperature-sensitive semi-dwarf (TSSD) cultivar which might be useful as breeding stock. The genome of CN14 was sequenced and assembled de novo using single-molecule real-time sequencing and chromosome conformation capture assembly. A high-quality genome was assembled and annotated, with 228.82 Mb mapped to eight chromosomes. Eighty-six re-sequenced F1 individuals and 334 previously re-sequenced accessions were used to identify candidate genes controlling TSSD and flower type and size. An aquaporin tonoplast intrinsic protein (PpTIP2) was a strong candidate gene for control of TSSD. Sequence variations in the upstream regulatory region of PpTIP2 correlated with different transcriptional activity at different temperatures. PpB3-1, a candidate gene for flower type (SH) and flower size, contributed to petal development and promoted petal enlargement. The locus of another 12 agronomic traits was identified through genome-wide association study. Most of these loci exhibited consistent and precise association signals, except for flesh texture and flesh adhesion. A 6015-bp insertion in exon 3 and a 26-bp insertion upstream of PpMYB25 were associated with fruit hairless. Along with a 70.5-Kb gap at the F-M locus in CN14, another two new alleles were identified in peach accessions. Our findings will not only promote genomic research and agronomic breeding in peach but also provide a foundation for the peach pan-genome.

Phenylalanine 4-Hydroxylase Contributes to Endophytic Bacterium Pseudomonas fluorescens' Melatonin Biosynthesis.

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

Genome-wide identification of HSF family in peach and functional analysis of PpHSF5 involvement in root and aerial organ development.

BACKGROUND: Heat shock factors (HSFs) play important roles during normal plant growth and development and when plants respond to diverse stressors. Although most studies have focused on the involvement of HSFs in the response to abiotic stresses, especially in model plants, there is little research on their participation in plant growth and development or on the HSF (PpHSF) gene family in peach (Prunus persica). METHODS: DBD (PF00447), the HSF characteristic domain, was used to search the peach genome and identify PpHSFs. Phylogenetic, multiple alignment and motif analyses were conducted using MEGA 6.0, ClustalW and MEME, respectively. The function of PpHSF5 was confirmed by overexpression of PpHSF5 into Arabidopsis. RESULTS: Eighteen PpHSF genes were identified within the peach genome. The PpHSF genes were nonuniformly distributed on the peach chromosomes. Seventeen of the PpHSFs (94.4%) contained one or two introns, except PpHSF18, which contained three introns. The in silico-translated PpHSFs were classified into three classes (PpHSFA, PpHSFB and PpHSFC) based on multiple alignment, motif analysis and phylogenetic comparison with HSFs from Arabidopsis thaliana and Oryza sativa. Dispersed gene duplication (DSD at 67%) mainly contributed to HSF gene family expansion in peach. Promoter analysis showed that the most common cis-elements were the MYB (abiotic stress response), ABRE (ABA-responsive) and MYC (dehydration-responsive) elements. Transcript profiling of 18 PpHSFs showed that the expression trend of PpHSF5 was consistent with shoot length changes in the cultivar 'Zhongyoutao 14'. Further analysis of the PpHSF5 was conducted in 5-year-old peach trees, Nicotiana benthamiana and Arabidopsis thaliana, respectively. Tissue-specific expression analysis showed that PpHSF5 was expressed predominantly in young vegetative organs (leaf and apex). Subcellular localization revealed that PpHSF5 was located in the nucleus in N. benthamiana cells. Two transgenic Arabidopsis lines were obtained that overexpressed PpHSF5. The root length and the number of lateral roots in the transgenic seedlings were significantly less than in WT seedlings and after cultivation for three weeks. The transgenic rosettes were smaller than those of the WT at 2-3 weeks. The two transgenic lines exhibited a dwarf phenotype three weeks after transplanting, although there was no significant difference in the number of internodes. Moreover, the PpHSF5-OE lines exhibited enhanced thermotolerance. These results indicated that PpHSF5 might be act as a suppresser of growth and development of root and aerial organs.

Functional Analysis of the Gibberellin 2-oxidase Gene Family in Peach.

Peach (Prunus persica L. Batsch) trees grow vigorously and are subject to intense pruning during orchard cultivation. Reducing the levels of endogenous gibberellins (GAs) represents an effective method for controlling branch growth. Gibberellin 2-oxidases (GA2oxs) deactivate bioactive GAs, but little is known about the GA2ox gene family in peach. In this study, we identified seven PpGA2ox genes in the peach genome, which were clustered into three subgroups: C19-GA2ox-I, C19-GA2ox-II, and C20-GA2ox-I. Overexpressing representative genes from the three subgroups, PpGA2ox-1, PpGA2ox-5, and PpGA2ox-2, in tobacco resulted in dwarf plants with shorter stems and smaller leaves than the wild type. An analysis of the GA metabolic profiles of the transgenic plants showed that PpGA2ox-5 (a member of subgroup C19-GA2ox-II) is simultaneously active against both C19-GAs and C20-GAs,which implied that C19-GA2ox-II enzymes represent intermediates of C19-GA2oxs and C20-GA2oxs. Exogenous GA3 treatment of shoot tips activated the expression of all seven PpGA2ox genes, with different response times: the C 19-GA2ox genes were transcriptionally activated more rapidly than the C20-GA2ox genes. GA metabolic profile analysis suggested that C20-GA2ox depletes GA levels more broadly than C19-GA2ox. These results suggest that the PpGA2ox gene family is responsible for fine-tuning endogenous GA levels in peach. Our findings provide a theoretical basis for appropriately controlling the vigorous growth of peach trees.