FvUVI4 inhibits cell division and cell expansion to modulate fruit development in Fragaria vesca.

Fruit development is mainly regulated by cell division and expansion. As a negative regulator of the anaphase-promoting complex/cyclosome, UVI4 plays important roles in plant growth and development via coordinating cell cycle. However, currently there is no report on UVI4's functions in regulating fruit development in strawberry. Here, Fragaria vesca homolog FvUVI4 is identified and localizes in the nucleus. FvUVI4 has high gene expression in roots, leaves, flower, buds and green fruits, and low expression in petiole, stem, white and yellow fruit. Fruit development of F. vesca 'Hawaii4' is regulated by endoreduplication, and the expression of FvUVI4 is negatively correlated with fruit cell size. Overexpression of FvUVI4 inhibits endoreduplication of leaves, flowers and fruits in both Arabidopsis and F. vesca 'Hawaii4', thereby limiting cell expansion and decreasing cell area. Overexpression of FvUVI4 also inhibits mitotic cell cycle leading to decreased cell number, and ultimately affects the growth of leaves, petals and seeds or fruits. Arabidopsis uvi4 mutants obtained via CRISPR-Cas9 technology display opposite growth phenotypes to Arabidopsis and F. vesca 'Hawaii4' overexpression lines, which can be restored by overexpression of FvUVI4 in Arabidopsis uvi4 mutants. In conclusion, our study indicates that FvUVI4 inhibits cell expansion and cell division to modulate receptacle development in woodland strawberry.

Identification and functional characterization of AcMYB113 in anthocyanin metabolism of Aesculus chinensis Bunge var. chinensis leaves.

Anthocyanins can be induced by environmental factors such as low-temperature and play essential roles in plant color formation. In this study, leaves of Aesculus chinensis Bunge var. chinensis with different colors under natural low-temperature in autumn were collected and grouped into green leaf (GL) and red leaf (RL). To reveal the underlying mechanism of color formation in RL, a combined analysis of the metabolome and transcriptome was conducted with GL and RL. Metabolic analyses revealed that total anthocyanin content and primary anthocyanin components were increased RL relative to GL and cyanidin was the main anthocyanin compound in RL. Transcriptome analysis provided a total of 18720 differentially expressed genes (DEGs), of which 9150 DEGs were upregulated and 9570 DEGs were downregulated in RL relative to GL. KEGG analysis showed that DEGs were mainly enriched in flavonoid biosynthesis, phenylalanine metabolism, and phenylpropanoid biosynthesis. Furthermore, co-expression network analysis indicated that 56 AcMYB transcription factors were highly expressed in RL compared with GL, among which AcMYB113 (an R2R3-MYB TF) had a strong correlation with anthocyanins. Overexpression of AcMYB113 in apple resulted in dark-purple transgenic calluses. In addition, the transient expression experiment showed that AcMYB113 enhanced anthocyanin synthesis by activating pathways of anthocyanin biosynthesis in leaves of Aesculus chinensis Bunge var. chinensis. Taken together, our findings reveal new insights into the molecular mechanism of anthocyanin accumulation in RL and provide candidate genes for the breeding of anthocyanin-rich cultivars.

Volatile secondary metabolome and transcriptome analysis reveals distinct regulation mechanism of aroma biosynthesis in Syringa oblata and S. vulgaris.

Lilacs have high ornamental value due to their strong aroma. However, the molecular regulatory mechanisms of aroma biosynthesis and metabolism in lilac were largely unclear. In this study, two varieties with distinct aroma, Syringa oblata 'Zi Kui' (faint aroma) and Syringa vulgaris 'Li Fei' (strong aroma), were used for exploring the regulation mechanism of aroma difference. Via GC-MS analysis, a total of 43 volatile components were identified. Terpene volatiles was the most abundant volatiles constituting the aroma of two varieties. Notably, 3 volatile secondary metabolites were unique in 'Zi Kui' and 30 volatile secondary metabolites were unique in 'Li Fei'. Then, a transcriptome analysis was performed to clarify the regulation mechanism of aroma metabolism difference between these two varieties, and identified 6411 differentially expressed genes (DEGs). Interestingly, ubiquinone and other terpenoid-quinone biosynthesis genes were significantly enriched in DEGs. We further conducted a correlation analysis between the volatile metabolome and transcriptome and found that TPS, GGPPS, and HMGS genes might be the key contributors to the differences in floral fragrance composition between the two lilac varieties. Our study improves the understanding in the regulation mechanism of Lilac aroma and would help improve the aroma of ornamental crops by metabolic engineering.

Association study of SNP locus for color related traits in herbaceous peony (Paeonia lactiflora Pall.) using SLAF-seq.

Paeonia lactiflora Pall. (P. lactiflora) is a famous ornamental plant with showy and colorful flowers that has been domesticated in China for 4,000 years. However, the genetic basis of phenotypic variation and genealogical relationships in P. lactiflora population is poorly understood due to limited genetic information, which brings about bottlenecks in the application of effective and efficient breeding strategies. Understanding the genetic basis of color-related traits is essential for improving flower color by marker-assisted selection (MAS). In this study, a high throughput sequencing of 99 diploid P. lactiflora accessions via specific-locus amplified fragment sequencing (SLAF-seq) technology was performed. In total, 4,383,645 SLAF tags were developed from 99 P. lactiflora accessions with an average sequencing depth of 20.81 for each SLAF tag. A total of 2,954,574 single nucleotide polymorphisms (SNPs) were identified from all SLAF tags. The population structure and phylogenetic analysis showed that P. lactiflora population used in this study could be divided into six divergent groups. Through association study using Mixed linear model (MLM), we further identified 40 SNPs that were significantly positively associated with petal color. Moreover, a derived cleaved amplified polymorphism (dCAPS) marker that was designed based on the SLAF tag 270512F co-segregated with flower colors in P. lactiflora population. Taken together, our results provide valuable insights into the application of MAS in P. lactiflora breeding programs.

H2 S improves salt-stress recovery via organic acid turn-over in apple seedlings.

Signalling roles of hydrogen sulphide (H2 S) in stress biology are widely reported but not sufficiently established to urge its use in agronomic practice. Our lack of quantitative understanding of the metabolic rewiring in H2 S signalling makes it difficult to elucidate its functions in stress tolerance on the biochemical level. Here, Malus hupehensis Rehd. var. pingyiensis seedlings were first treated with salt stress for 2 weeks and then treated with four different concentrations of NaHS. Through vigorous investigations, including phenotypic analysis, 13 C transient labelling and targeted metabolic and transcriptomic analysis, for the first time in the seedlings of a woody fruit crop, we found out that H2 S recycles fixed carbons through glycolysis and tricarboxylic acid cycle to inhibit the futile accumulation of carbohydrates, to maintain an efficient CO2 assimilation, to keep a balanced starch metabolism, to produce sufficient H2 O2 , to maintain malate/γ-aminobutyric acid homeostasis via an H2 O2 -induced anion channel (aluminium-activated malate transporter) and eventually to improve salt-stress recovery. Our results systematically demonstrate the vital roles of central carbon metabolism in H2 S signalling and clarify the mode of action of H2 S in apple seedlings. We conclude that H2 S signalling interacts with central carbon metabolism in a bottom-up manner to recover plant growth after salt stress.

Auxin alters sodium ion accumulation and nutrient accumulation by playing protective role in salinity challenged strawberry.

High salinity in soil affects the strawberry production and fruit quality. Auxin-primed plants have enhanced responses to soil salinization. In this study, we report that exogenous application of IAA can partially relieve stress responses of strawberry seedlings. Cytological analysis showed that the ultrastructure of root tip and leaf cells in strawberry seedlings were altered under high salinity condition, which was partially recovered after the application of IAA. The study showed that the ultrastructure of root tip and leaf cells in strawberry seedlings were altered under salt stress condition, which was partially recovered after the application of IAA. Exogenous IAA ameliorated deleterious effects on seedling growth under salinity were attributed to accelerated Na+ fluxes, decreased the contents of Na+ to maintain the ion homeostasis, protect root growth, and promote the absorption of nutrients for improved photosynthetic efficiency in strawberry.

Comparative N-glycoproteome analysis provides novel insights into the regulation mechanism in tomato (solanum lycopersicum L.) During fruit ripening process.

Protein N-glycosylation plays key roles in protein folding, stability, solubility, biogenesis, and enzyme activity. Tomato (Solanum lycopersicum L.) is an important vegetable crop with abundant nutritional value, and the formation of tomato fruit qualities primarily occurs in the fruit ripening process. However, a large number of N-glycosylation-mediated mechanisms in regulating tomato fruit ripening have not been elucidated to date. In this study, western blot assays showed that the extents of mature N-glycoproteins were differentially expressed in mature green fruits (fruit start ripening) and ripe fruits (fruit stop ripening). Next, through performing a comparative N-glycoproteome analysis strategy, a total of 553 N-glycosites from 363 N-glycoproteins were identified in mature green fruits compared with ripe fruits. Among them, 252 N-glycosites from 191 N-glycoproteins were differentially expressed in mature green fruits compared with ripe fruits. The differentially expressed N-glycoproteins were mainly located in the chloroplast (30 %) and cytoplasm (16 %). Gene Ontology (GO) analysis showed that these N-glycoproteins were involved in various biological processes, cellular components and molecular functions. These N-glycoproteins participate in biological processes, such as metabolic processes, cellular processes and single-organism processes. These N-glycoproteins are also cellular components in biological process cells, membranes and organelles and have different molecular functions, such as catalytic activity and binding. Notably, these N-glycoproteins were enriched in starch and sucrose metabolism and galactose metabolism by KEGG pathway analysis. This community resource regarding N-glycoproteins is the first large-scale N-glycoproteome during plant fruit ripening. This study will contribute to understanding the function of N-glycosylation in regulating plant fruit ripening.

A synthetic peptide encoded by a random DNA sequence inhibits discrete red light responses.

We have identified a synthetic peptide that interrupts discrete aspects of seedling development under red light. Previous reports have demonstrated that plants transformed with random DNA sequences produce synthetic peptides that affect plant biology. In this report, one specific peptide is characterized that inhibits discrete aspects of red light-mediated photomorphogenic development in Arabidopsis thaliana . Seedlings expressing the PEP6-32 peptide presented longer hypocotyls and diminished cotyledon expansion when grown under red light. Other red light-mediated seedling processes such as induction of Lhcb (cab) transcripts or loss of vertical growth remained unaffected. Long-term responses to red light in PEP6-32 expressing plants, such as repression of flowering time, did not show defects in red light signaling or integration. A synthesized peptide applied exogenously induced the long-hypocotyl phenotype under red light in non-transformed seedlings. The results indicate that the PEP6-32 peptide causes discrete cell expansion abnormalities during early seedling development in red light that mimic weak phyB alleles, yet only in some aspects of seedling photomorphogenesis. The findings demonstrate that new chemistries derived from random peptide expression can modulate specific facets of plant growth and development.

MOS1 functions closely with TCP transcription factors to modulate immunity and cell cycle in Arabidopsis.

Emerging evidence indicates a close connection between cell-cycle progression and the plant immune responses. In Arabidopsis, MODIFIER OF snc1-1 (MOS1) modulates a number of processes including endoreduplication and plant disease resistance, but the molecular mechanism underlying this modulation was not fully understood. Here, we provide biochemical and genetic evidence that TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors TCP15 and its homologues are mediators of MOS1 function in the immune response and are likely to be also involved in cell-cycle control. MOS1 and TCP proteins have a direct physical interaction. They both bind to the promoter of the immune receptor gene SUPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) and modulate its expression and consequently immune responses. MOS1 and TCP15 both affect the expression of cell-cycle genes D-type CYCLIN 3;1 (CYCD3;1), which may mediate the MOS1 function in cell-cycle modulation. In addition, CYCD3;1 overexpression upregulates immune responses, and SNC1 expression. This study investigated and revealed a role for MOS1 in transcriptional regulation through TCP15 and its homologues. This finding suggests the coordination of cell-cycle progression and plant immune responses at multiple levels.

Identification of a methyltransferase catalyzing the final step of methyl anthranilate synthesis in cultivated strawberry.

BACKGROUND: Methyl anthranilate (MA) contributes an attractive fruity note to the complex flavor and aroma of strawberry (Fragaria spp.), yet it is rare in modern cultivars. The genetic basis for its biosynthesis has not been elucidated. Understanding the specific genes required for its synthesis could allow  the development of gene/allele-specific molecular markers to speed breeding of flavorful strawberries. RESULTS: Ripe fruits from individuals in an F1 population resulting from a cross between a MA producer and a non-producer were examined using a bulk-segregant transcriptome approach. MA producer and non-producer transcriptomes were compared, revealing five candidate transcripts that strictly co-segregated with MA production. One candidate encodes an annotated methyltransferase. MA levels are lower when this transcript is suppressed with RNAi, and bacterial cultures expressing the protein produced MA in the presence of anthranilic acid. Frozen fruit powders reconstituted with anthranilic acid and a methyl donor produced MA only if the transcript was detected in the fruit powder. A DNA-based molecular marker was developed that segregates with the MA-producing gene variant. CONCLUSIONS: These analyses indicate that the methyltransferase, now noted ANTHRANILIC ACID METHYL TRANSFERASE (FanAAMT), mediates the ultimate step of MA production in cultivated strawberry. Identification of this gene and its associated molecular marker may hasten breeding efforts to introduce this important volatile into modern cultivars.

Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides.

The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds.

Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system.

Plants and animals detect the presence of potential pathogens through the perception of conserved microbial patterns by cell surface receptors. Certain solanaceous plants, including tomato, potato and pepper, detect flgII-28, a region of bacterial flagellin that is distinct from that perceived by the well-characterized FLAGELLIN-SENSING 2 receptor. Here we identify and characterize the receptor responsible for this recognition in tomato, called FLAGELLIN-SENSING 3. This receptor binds flgII-28 and enhances immune responses leading to a reduction in bacterial colonization of leaf tissues. Further characterization of FLS3 and its signalling pathway could provide new insights into the plant immune system and transfer of the receptor to other crop plants offers the potential of enhancing resistance to bacterial pathogens that have evolved to evade FLS2-mediated immunity.

Identification of a Candidate Gene in Solanum habrochaites for Resistance to a Race 1 Strain of Pseudomonas syringae pv. tomato.

Bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst) is a persistent problem on tomato (Solanum lycopersicum L.). Resistance against race 0 Pst strains is conferred by the Pto protein, which recognizes either of two pathogen effectors: AvrPto or AvrPtoB. However, current tomato varieties do not have resistance to the increasingly common race 1 strains, which lack these effectors. We identified accessions of Solanum habrochaites S. Knapp & D. M. Spooner that are resistant to the race 1 strain T1. Genome sequence comparisons of T1 and two Pst strains that are virulent on these accessions suggested that known microbe-associated molecular patterns (MAMPs) or effectors are not involved in the resistance. We developed an F2 population from a cross between one T1-resistant accession, LA2109, and a susceptible tomato cultivar to investigate the genetic basis of this resistance. Linkage analysis using whole-genome sequence of 58 F2 plants identified quantitative trait loci (QTL), qRph1, in a 5.8-Mb region on chromosome 2, and qRph2, in a 52.4-Mb region on chromosome 8, which account for 24 and 26% of the phenotypic variability, respectively. High-resolution mapping of qRph1 confirmed it contributed to T1 resistance and delimited it to a 1060-kb region containing 139 genes, including three encoding receptor-like proteins (RLPs) and 17 encoding receptor-like protein kinases (RLKs). One RLK gene, Solyc02g072470, is a promising candidate for qRph1, as it is highly expressed in LA2109 and induced on treatment with MAMPs. qRph1 might be useful for enhancing resistance to race 1 strains and its future characterization could provide insights into the plant immune system.

Endopolyploidization and flowering time are antagonistically regulated by checkpoint component MAD1 and immunity modulator MOS1.

The spindle assembly checkpoint complex (SAC) is essential for quality control during mitosis in yeast and animals. However, its function in plants is not well understood. Here we show that MAD1, an Arabidopsis SAC component, is involved in endopolyploidization and flowering time via genetic interaction with MOS1, a negative regulator of plant immunity. MOS1 is found to interact with MAD2, another SAC component, and promote flowering and inhibit endopolyploidization, but this function is antagonized by MAD1. Furthermore, MAD1 and MOS1 both interact with SUF4, a transcription factor regulating the expression of the flowering time gene FLC. These findings reveal MOS1, MAD1 and SUF4 as regulators of endopolyploidization and flowering time and suggest an involvement of cell cycle control in the timing of reproductive transition.

Interaction of CPR5 with cell cycle regulators UVI4 and OSD1 in Arabidopsis.

The impact of cell cycle on plant immunity was indicated by the enhancement of disease resistance with overexpressing OSD1 and UVI4 genes that are negative regulators of cell cycle controller APC (anaphase promoting complex). CPR5 is another gene that is implicated in cell cycle regulation and plant immunity, but its mode of action is not known. Here we report the analysis of genetic requirement for the function of UVI4 and OSD1 in cell cycle progression control and in particular the involvement of CPR5 in this regulation. We show that the APC activator CCS52A1 partially mediates the function of OSD1 and UVI4 in female gametophyte development. We found that the cpr5 mutation suppresses the endoreduplication defect in the uvi4 single mutant and partially rescued the gametophyte development defect in the osd1 uvi4 double mutant while the uvi4 mutation enhances the cpr5 defects in trichome branching and plant disease resistance. In addition, cyclin B1 genes CYCB1;1, CYCB1;2, and CYCB1;4 are upregulated in cpr5. Therefore, CPR5 has a large role in cell cycle regulation and this role has a complex interaction with that of UVI4 and OSD1. This study further indicates an intrinsic link between plant defense responses and cell cycle progression.

Perturbation of cell cycle regulation triggers plant immune response via activation of disease resistance genes.

The Arabidopsis gene OSD1 (Omission of the Second Division) and its homolog UVI4 (UV-B-Insensitive 4) are negative regulators of anaphase-promoting complex/cyclosome (APC/C), a multisubunit ubiquitin E3 ligase that regulates the progression of cell cycles. Here we report the isolation of an activation tagging allele of OSD1 as an enhancer of a mutant of BON1 (BONZAI1), a negative regulator of plant immunity. Overexpression of OSD1 and UVI4 each leads to enhanced immunity to a bacterial pathogen, which is associated with increased expression of disease resistance (R) genes similar to the animal NOD1 receptor-like immune receptor genes. In addition, the reduction of function of one subunit of the APC complex APC10 exhibited a similar phenotype to that of overexpression of OSD1 or UVI4, indicating that altered APC function induces immune responses. Enhanced immune response induced by OSD1 overexpression is dependent on CYCB1;1, which is a degradation target of APC/C. It is also associated with up-regulation of R genes and is dependent on the R gene SNC1 (Suppressor of npr1-1, constitutive 1). Taken together, our findings reveal an unexpected link between cell cycle progression and plant immunity, suggesting that cell cycle misregulation could have an impact on expression of genes, including R genes, in plant immunity.

The F-box protein CPR1/CPR30 negatively regulates R protein SNC1 accumulation.

Disease resistance (R) proteins, as central regulators of plant immunity, are tightly regulated for effective defense responses and to prevent constitutive defense activation under non-pathogenic conditions. Here we report the identification of an F-box protein CPR1/CPR30 as a negative regulator of an R protein SNC1 likely through SCF (Skp1-cullin-F-box) mediated protein degradation. The cpr1-2 (cpr30-1) loss-of-function mutant has constitutive defense responses, and it interacts synergistically with a gain-of function mutant snc1-1 and a bon1-1 mutant where SNC1 is upregulated. The loss of SNC1 function suppresses the mutant phenotypes of cpr1-2 and cpr1-2 bon1-1, while overexpression of CPR1 rescues mutant phenotypes of both bon1-1 and snc1-1. Furthermore, the amount of SNC1 protein is upregulated in the cpr1-2 mutant and down-regulated when CPR1 is overexpressed. The regulation of SNC1 by CPR1 is dependent on the 26S proteosome as a protease inhibitor MG132 stabilizes SNC1 and reverses the effect of CPR1 on SNC1. Interestingly, CPR1 is induced after infection of both virulent and avirulent pathogens similarly to the other negative defense regulator BON1. Thus, this study reveals a new mechanism in R protein regulation likely through protein degradation and suggests negative regulation as a critical component in fine control of plant immunity.

Analysis of temperature modulation of plant defense against biotrophic microbes.

Plant-pathogen interactions are known to be affected by environmental factors including temperature; however, the temperature effects have not been systematically studied in plant disease resistance. Here, we characterized the effects of a moderate increase in temperature on resistance to bacterial pathogen Pseudomonas syringae and two viral elicitors in Arabidopsis thaliana and Nicotiana benthamiana. Both the basal and the resistance (R) gene-mediated defense responses to Pseudomonas syringae are found to be inhibited by a moderately high temperature, and hypersensitive responses induced by R genes against two viruses are also reduced by an increase of temperature. These indicate that temperature modulation of defense responses to biotrophic and hemibiotrophic pathogens might be a general phenomenon. We further investigated the roles of two small signaling molecules, salicylic acid and jasmonic acid, as well as two defense regulators, EDS1 and PAD4, in this temperature modulation. These components, though modulated by temperature or involved in temperature regulation or both, are not themselves determinants of temperature sensitivity in the defense responses analyzed. The inhibition of plant defense response by a moderately high temperature may thus be mediated by other defense signaling components or a combination of multiple factors.

The ABI2-dependent abscisic acid signalling controls HrpN-induced drought tolerance in Arabidopsis.

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.