Two B-box proteins, PavBBX6/9, positively regulate light-induced anthocyanin accumulation in sweet cherry.

Anthocyanin production in bicolored sweet cherry (Prunus avium cv. Rainier) fruit is induced by light exposure, leading to red coloration. The phytohormone abscisic acid (ABA) is essential for this process, but the regulatory relationships that link light and ABA with anthocyanin-associated coloration are currently unclear. In this study, we determined that light treatment of bicolored sweet cherry fruit increased anthocyanin accumulation and induced ABA production and that ABA participates in light-modulated anthocyanin accumulation in bicolored sweet cherry. Two B-box (BBX) genes, PavBBX6/9, were highly induced by light and ABA treatments, as was anthocyanin accumulation. The ectopic expression of PavBBX6 or PavBBX9 in Arabidopsis (Arabidopsis thaliana) increased anthocyanin biosynthesis and ABA accumulation. Overexpressing PavBBX6 or PavBBX9 in sweet cherry calli also enhanced light-induced anthocyanin biosynthesis and ABA accumulation. Additionally, transient overexpression of PavBBX6 or PavBBX9 in sweet cherry peel increased anthocyanin and ABA contents, whereas silencing either gene had the opposite effects. PavBBX6 and PavBBX9 directly bound to the G-box elements in the promoter of UDP glucose-flavonoid-3-O-glycosyltransferase (PavUFGT), a key gene for anthocyanin biosynthesis, and 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1), a key gene for ABA biosynthesis, and enhanced their activities. These results suggest that PavBBX6 and PavBBX9 positively regulate light-induced anthocyanin and ABA biosynthesis by promoting PavUFGT and PavNCED1 expression, respectively. Our study provides insights into the relationship between the light-induced ABA biosynthetic pathway and anthocyanin accumulation in bicolored sweet cherry fruit.

MicroRNA156ab regulates apple plant growth and drought tolerance by targeting transcription factor MsSPL13.

Drought stress substantially reduces the productivity of apple plants and severely restricts the development of apple industry. Malus sieversii, wild apples with excellent drought resistance, is a valuable wild resource for a rootstock improvement of cultivated apple (Malus domestica). miRNAs and their targets play essential roles in plant growth and stress responses, but their roles in drought stress responses in apple are unknown. Here, we demonstrate that microRNA156ab is upregulated in M. sieversii in response to drought stress. Overexpressing msi-miR156ab promoted auxin accumulation, maintained the growth of apple plants, and increased plant resistance to osmotic stress. Antioxidant enzyme activities and proline contents were also increased in miR156ab-OE transgenic apple lines, which improved drought resistance. The squamosa promoter binding protein-like transcription factor MsSPL13 is the target of msi-miR156ab, as demonstrated by 5'-RACE and dual luciferase assays. Heterologous expression of MsSPL13 decreased auxin contents and inhibited growth in Arabidopsis (Arabidopsis thaliana) under normal and stress conditions. The activities of antioxidant enzymes were also suppressed in MsSPL13-OE transgenic Arabidopsis, reducing drought resistance. We showed that MsSPL13 regulates the expression of the auxin-related genes MsYUCCA5, PIN-FORMED7 (MsPIN7), and Gretchen Hagen3-5 (MsGH3-5) by binding to the GTAC cis-elements in their promoters, thereby regulating auxin metabolism. Finally, we demonstrated that the miR156ab-SPL13 module is involved in mediating the difference in auxin metabolism and stress responses between M. sieversii and M26 (M. domestica) rootstocks. Overall, these findings reveal that the miR156ab-SPL13 module enhances drought stress tolerance in apples by regulating auxin metabolism and antioxidant enzyme activities.

Abscisic acid-responsive transcription factors PavDof2/6/15 mediate fruit softening in sweet cherry.

Softening is a key step during fruit ripening that is modulated by the interplay between multiple phytohormones. The antagonistic action of abscisic acid (ABA) and auxin determines the rate of fruit ripening and softening. However, the transcription factors that integrate ABA and auxin signals to regulate fruit softening remain to be determined. In this study, we identified several DNA-binding with One Finger (Dof) transcription factors essential for ABA-promoted fruit softening, based on transcriptome analysis of two sweet cherry (Prunus avium L.) varieties with different fruit firmness. We show that PavDof6 directly binds to the promoters of genes encoding cell wall-modifying enzymes to activate their transcription, while PavDof2/15 directly repress their transcription. Transient overexpression of PavDof6 and PavDof2/15 in sweet cherry fruits resulted in precocious and delayed softening, respectively. In addition, we show that the auxin response factor PavARF8, the expression of whose encoding gene is repressed by ABA, activates PavDof2/15 transcription. Furthermore, PavDof2/6/15 and PavARF8 directly bind to the 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1) promoter and regulate its expression, forming a feedback mechanism for ABA-mediated fruit softening. These findings unveil the physiological framework of fruit softening and establish a direct functional link between the ABA-PavARF8-PavDofs module and cell-wall-modifying genes in mediating fruit softening.

Genome-Wide Identification of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Sweet Cherry Fruit.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play important regulatory roles during plant growth and development, fruit ripening, inflorescence branching, and biotic and abiotic stresses. However, there have been no identification or systematic studies of the SPL gene family in the sweet cherry. In this study, 12 SPL genes were identified in the sweet cherry reference genome, which were distributed over 6 chromosomes and classified into six groups according to phylogenetic relationships with other SPL gene families. Nine PavSPLs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, which implied that they were important regulators during fruit development and ripening. The expression patterns of PavSPL genes under ABA, GA, and MeJA treatments showed that the PavSPLs were involved in the process of fruit ripening. A subcellular localization experiment proved that PavSPL4 and PavSPL7 proteins were localized in the nucleus. The genome-wide identification of the SPL gene family provided new insights while establishing an important foundation for sweet cherry studies.

Over-Expression of an R2R3 MYB Gene, MdMYB108L, Enhances Tolerance to Salt Stress in Transgenic Plants.

Plants are affected by various abiotic stresses during their growth and development. In plants, MYB transcription factors are involved in various physiological and biochemical processes, including biotic and abiotic stress responses. In this study, we functionally analyzed MdMYB108L. We examined the transcriptional activity of MdMYB108L under salt stress and determined that the N-terminal domain of MdMYB108L, which was significantly induced under salt stress, has transcriptional activity. MdMYB108L overexpression increased the germination rate, main root length, and the antioxidant activity of catalase and peroxidase in transgenic Arabidopsisthaliana seeds, while reducing reactive oxygen species (ROS) accumulation. MdMYB108L overexpression also increased the photosynthetic capacity of hairy root tissue (leaves) under salt stress. In addition, the MdMYB108L transcription factor bound to the MdNHX1 promoter positively regulated the transcription of the salt tolerance gene MdNHX1 in apples, improving the salt stress tolerance of transgenic plants. These findings have implications for improving the agricultural yields of apple trees under salt stress.

Genome-Wide Identification of the Xyloglucan endotransglucosylase/Hydrolase (XTH) and Polygalacturonase (PG) Genes and Characterization of Their Role in Fruit Softening of Sweet Cherry.

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes.

The Nicotiana tabacum L. major latex protein-like protein 423 (NtMLP423) positively regulates drought tolerance by ABA-dependent pathway.

BACKGROUND: Drought stress is an environmental factor that limits plant growth and reproduction. Little research has been conducted to investigate the MLP gene in tobacco. Here, NtMLP423 was isolated and identified, and its role in drought stress was studied. RESULTS: Overexpression of NtMLP423 improved tolerance to drought stress in tobacco, as determined by physiological analyses of water loss efficiency, reactive oxygen species levels, malondialdehyde content, and levels of osmotic regulatory substances. Overexpression of NtMLP423 in transgenic plants led to greater sensitivity to abscisic acid (ABA)-mediated seed germination and ABA-induced stomatal closure. NtMLP423 also regulated drought tolerance by increasing the levels of ABA under conditions of drought stress. Our study showed that the transcription level of ABA synthetic genes also increased. Overexpression of NtMLP423 reduced membrane damage and ROS accumulation and increased the expression of stress-related genes under drought stress. We also found that NtWRKY71 regulated the transcription of NtMLP423 to improve drought tolerance. CONCLUSIONS: Our results indicated that NtMLP423-overexpressing increased drought tolerance in tobacco via the ABA pathway.

Multiplex-PCR method application to identify duck blood and its adulterated varieties.

This study applied and validated the Multiplex-PCR method to identify the authenticity of duck blood and four common adulterated animal blood varieties. To this end, the genomic DNAs of duck blood and its counterfeit products were extracted using an efficient high-throughput extraction method. Specific primers were designed using the cytochrome b gene. The reaction system and conditions of a multiplex (namely, Five-plex) PCR were optimized, and the proposed methodology was verified, proving its good specificity, repeatability, and sensitivity. The Five-plex PCR system detected nine duck blood samples sold in the local market, revealing the adulteration of duck blood products. The Multiplex-PCR system can accurately and quickly detect adulterated animal blood in duck blood products, effectively finding counterfeits and identifying the authenticity of genuine duck blood products.

Overexpression of major latex protein 423 (NtMLP423) enhances the chilling stress tolerance in Nicotiana tabacum.

Chilling stress impedes plant growth and hinders crop development and productivity. In this study, we identified the major latex protein (MLP) in tobacco (NtMLP423) and examined its roles in chilling resistance. NtMLP423 expression was considerably upregulated in response to chilling stress. NtMLP423 function was assessed and compared in plants with overexpression and antisense characteristics. Under chilling stress, plants with overexpression characteristics grew better than wild-type and antisense plants. NtMLP423 overexpression reduced membrane lipid damage, increased antioxidant enzyme activity, and reduced reactive oxygen species (ROS) accumulation under chilling stress. Here, we screened for the first time the upstream transcription factor NtMYB108, which regulates NtMLP423 expression under chilling stress. The NtMYB108 transcription factor directly binds to the NtMLP423 promoter and improves NtMLP423 resistance to chilling stress. Subjecting NtMYB018 to virus-induced gene silencing reduced chilling stress tolerance. Overall, NtMLP423 overexpression enhances chilling stress tolerance, while its suppression has the opposite effect.

miR164g-MsNAC022 acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple.

Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line 'GL-3' (Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g-MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars.

Aloe vera gel coating aggravates superficial scald incidence in 'Starking' apples during low-temperature storage.

The effects of aloe vera (Aloe vera (L.) Burm. f.) gel treatment on the incidence of superficial scald in 'Starking' apples (Malus domestica Borkh. Var. Starking) during cold storage were studied. Apples were harvested at the pre-climacteric stage and treated with aloe vera gel. The treatment increased malondialdehyde content and membrane lipid damage. Furthermore, it inhibited the release of ethylene at the early stage but increased it in the later stage. The expression level of ACC synthase 1 (MdACS1) also increased, and the antioxidant capacity in apples, particularly, catalase, peroxidase, and superoxide dismutase activities, all decreased, while concomitantly, the content of α-farnesene and its oxidation product, conjugated triene increased, thereby aggravating superficial scald incidence during storage at low temperature.