Overexpression of PavHIPP16 from Prunus avium enhances cold stress tolerance in transgenic tobacco.

BACKGROUND: The heavy metal-associated isoprenylated plant protein (HIPP) is an important regulatory element in response to abiotic stresses, especially playing a key role in low-temperature response. RESULTS: This study investigated the potential function of PavHIPP16 up-regulated in sweet cherry under cold stress by heterologous overexpression in tobacco. The results showed that the overexpression (OE) lines' growth state was better than wild type (WT), and the germination rate, root length, and fresh weight of OE lines were significantly higher than those of WT. In addition, the relative conductivity and malondialdehyde (MDA) content of the OE of tobacco under low-temperature treatment were substantially lower than those of WT. In contrast, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activities, hydrogen peroxide (H2O2), proline, soluble protein, and soluble sugar contents were significantly higher than those of WT. Yeast two-hybrid assay (Y2H) and luciferase complementation assay verified the interactions between PavbHLH106 and PavHIPP16, suggesting that these two proteins co-regulated the cold tolerance mechanism in plants. The research results indicated that the transgenic lines could perform better under low-temperature stress by increasing the antioxidant enzyme activity and osmoregulatory substance content of the transgenic plants. CONCLUSIONS: This study provides genetic resources for analyzing the biological functions of PavHIPPs, which is important for elucidating the mechanisms of cold resistance in sweet cherry.

Chinese cherry CpMYB44-CpSPDS2 module regulates spermidine content and florescence in tobacco.

The flower bud differentiation plays a crucial role in cherry yield and quality. In a preliminary study, we revealed the promotion of spermidine (Spd) in bud differentiation and quality. However, the molecular mechanism underlying Spd regulating cherry bud differentiation remains unclear. To address this research gap, we cloned CpSPDS2, a gene that encodes Spd synthase and is highly expressed in whole flowers and pistils of the Chinese cherry (cv. 'Manaohong'). Furthermore, an overexpression vector with this gene was constructed to transform tobacco plants. The findings demonstrated that transgenic lines exhibited higher Spd content, an earlier flowering time by 6 d, and more lateral buds and flowers than wild-type lines. Additionally, yeast one-hybrid assays and two-luciferase experiments confirmed that the R2R3-MYB transcription factor (CpMYB44) directly binds to and activates the CpSPDS2 promoter transcription. It is indicated that CpMYB44 promotes Spd accumulation via regulating CpSPDS2 expression, thus accelerating the flower growth. This research provides a basis for resolving the molecular mechanism of CpSPDS2 involved in cherry bud differentiation.

Overexpression of PavbHLH28 from Prunus avium enhances tolerance to cold stress in transgenic Arabidopsis.

BACKGROUND: The basic helix-loop-helix (bHLH) gene family is one of plants' largest transcription factor families. It plays an important role in regulating plant growth and abiotic stress response. RESULTS: In this study, we determined that the PavbHLH28 gene participated in cold resistance. The PavbHLH28 gene was located in the nucleus and could be induced by low temperature. Under the treatment of ABA, PEG, and GA3, the transcript level of PavbHLH28 was affected. At low temperature, overexpression of the PavbHLH28 gene enhanced the cold resistance of plants with higher proline content, lower electrolyte leakage (EL) and malondialdehyde (MDA) content. Compared with the WT plants, the transgenic plants accumulated fewer reactive oxygen species (ROS), and the activity and expression levels of antioxidant enzymes were significantly increased. The expression of proline synthesis enzyme genes was up-regulated, and the transcripts levels of degradation genes were significantly down-regulated. The transcripts abundance of the cold stressed-related genes in the C-repeat binding factor (CBF) pathway was not significantly different between WT plants and transgenic plants after cold stress. Moreover, the PavbHLH28 could directly bind to the POD2 gene promoter and promote its gene expression. CONCLUSIONS: Overall, PavbHLH28 enhanced the cold resistance of transgenic plants through a CBF-independent pathway, which may be partly related to ROS scavenging.

Copper Amine Oxidase (CuAO)-Mediated Polyamine Catabolism Plays Potential Roles in Sweet Cherry (Prunus avium L.) Fruit Development and Ripening.

Copper amine oxidases (CuAOs) play important roles in PA catabolism, plant growth and development, and abiotic stress response. In order to better understand how PA affects cherry fruit, four potential PavCuAO genes (PavCuAO1-PavCuAO4) that are dispersed over two chromosomes were identified in the sweet cherry genome. Based on phylogenetic analysis, they were classified into three subclasses. RNA-seq analysis showed that the PavCuAO genes were tissue-specific and mostly highly expressed in flowers and young leaves. Many cis-elements associated with phytohormones and stress responses were predicted in the 2 kb upstream region of the promoter. The PavCuAOs transcript levels were increased in response to abscisic acid (ABA) and gibberellin 3 (GA3) treatments, as well as abiotic stresses (NaCl, PEG, and cold). Quantitative fluorescence analysis and high-performance liquid chromatography confirmed that the Put content fell, and the PavCuAO4 mRNA level rose as the sweet cherry fruit ripened. After genetically transforming Arabidopsis with PavCuAO4, the Put content in transgenic plants decreased significantly, and the expression of the ABA synthesis gene NCED was also significantly increased. At the same time, excessive H2O2 was produced in PavCuAO4 transiently expressed tobacco leaves. The above results strongly proved that PavCuAO4 can decompose Put and may promote fruit ripening by increasing the content of ABA and H2O2 while suppressing total free PA levels in the fruit.

Overexpression of CpADC from Chinese Cherry (Cerasus pseudocerasus Lindl. 'Manaohong') Promotes the Ability of Response to Drought in Arabidopsis thaliana.

Polyamines (PA) play an important role in the growth, development and stress resistance of plants, and arginine decarboxylase (ADC) is one of the key enzymes in the biosynthetic pathway of polyamines. Previously, the transcriptional regulation of the 'Manaohong' cherry under the shelter covering was carried out, and the PA synthase-related genes, particularly the ADC gene, were differentially expressed as exposure to drought stress. However, the mechanisms of how ADC is involved in the response of cherry to abiotic stress (especially drought stress) are still unknown. In the present work, the full-length coding sequence of this gene was isolated and named CpADC. Bioinformatics analysis indicated that the coding sequence of CpADC was 2529 bp in length. Cluster analysis showed that CpADC had the highest homologies with those of sweet cherry (Prunus avium, XP_021806331) and peach (Prunus persica, XP_007200307). Subcellular localization detected that the CpADC was localized in the plant nucleus. The qPCR quantification showed that CpADC was differentially expressed in roots, stems, leaves, flower buds, flowers, and fruits at different periods. Drought stress treatments were applied to both wild-type (WT) and transgenic Arabidopsis lines, and relevant physiological indicators were measured, and the results showed that the putrescine content of transgenic Arabidopsis was higher than that of WT under high-temperature treatment. The results showed that the MDA content of WT was consistently higher than that of transgenic plants and that the degree of stress in WT was more severe than in transgenic Arabidopsis, indicating that transgenic CpADC was able to enhance the stress resistance of the plants. Both the transgenic and WT plants had significantly higher levels of proline in their leaves after the stress treatment than before, but the WT plant had lower levels of proline than that of transgenic Arabidopsis in both cases. This shows that the accumulation of proline in the transgenic plants was higher than that in the wild type under drought and high and low-temperature stress, suggesting that the transgenic plants are more stress tolerant than the WT. Taken together, our results reveal that, under drought stress, the increase in both expressions of CpADC gene and Put (putrescine) accumulation regulates the activity of ADC, the content of MDA and Pro to enhance the drought resistance of Arabidopsis thaliana.

Cross-Talk between Transcriptome Analysis and Dynamic Changes of Carbohydrates Identifies Stage-Specific Genes during the Flower Bud Differentiation Process of Chinese Cherry (Prunus pseudocerasus L.).

Flower bud differentiation is crucial to reproductive success in plants. In the present study, RNA-Seq and nutrients quantification were used to identify the stage-specific genes for flower bud differentiation with buds which characterize the marked change during flower bud formation from a widely grown Chinese cherry (Prunus pseudocerasus L.) cultivar 'Manaohong'. A KEGG enrichment analysis revealed that the sugar metabolism pathways dynamically changed. The gradually decreasing trend in the contents of total sugar, soluble sugar and protein implies that the differentiation was an energy-consuming process. Changes in the contents of D-glucose and sorbitol were conformed with the gene expression trends of bglX and SORD, respectively, which at least partially reflects a key role of the two substances in the transition from physiological to morphological differentiation. Further, the WRKY and SBP families were also significantly differentially expressed during the vegetative-to-reproductive transition. In addition, floral meristem identity genes, e.g., AP1, AP3, PI, AGL6, SEP1, LFY, and UFO demonstrate involvement in the specification of the petal and stamen primordia, and FPF1 might promote the onset of morphological differentiation. Conclusively, the available evidence justifies the involvement of sugar metabolism in the flower bud differentiation of Chinese cherry, and the uncovered candidate genes are beneficial to further elucidate flower bud differentiation in cherries.

The Roles of Auxin Biosynthesis YUCCA Gene Family in Plants.

Auxin plays essential roles in plant normal growth and development. The auxin signaling pathway relies on the auxin gradient within tissues and cells, which is facilitated by both local auxin biosynthesis and polar auxin transport (PAT). The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)/YUCCA (YUC) pathway is the most important and well-characterized pathway that plants deploy to produce auxin. YUCs function as flavin-containing monooxygenases (FMO) catalyzing the rate-limiting irreversible oxidative decarboxylation of indole-3-pyruvate acid (IPyA) to form indole-3-acetic acid (IAA). The spatiotemporal dynamic expression of different YUC gene members finely tunes the local auxin biosynthesis in plants, which contributes to plant development as well as environmental responses. In this review, the recent advances in the identification, evolution, molecular structures, and functions in plant development and stress response regarding the YUC gene family are addressed.

Physiological and Transcriptomic Changes during the Early Phases of Adventitious Root Formation in Mulberry Stem Hardwood Cuttings.

The initiation and induction of root primordia are of great importance for adventitious root (AR) formation in cutting propagation of horticultural and forestry crops. However, the underlying mechanisms orchestrating these early phases of AR formation remain largely unexplored. Here, we investigated the physiological and transcriptomic changes during the early AR phases in mulberry stem hardwood cuttings. The results showed that the concentrations of soluble proteins increased, whereas concentrations of soluble sugars and starch were decreased. Indole-3-acetic acid (IAA) and zeatin had a rapid transit peak at 6 h after planting (hAP) and declined thereafter. The activities of peroxidase and catalase persistently increased and indole-3-acetic acid oxidase was maintained at a higher stable level from 0 hAP, while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels. The comparative transcriptome identified 4276 common genes that were differentially regulated at -6, 0 and 54 hAP. They were separated into five clusters with distinct biological functions such as defense response and photosynthesis. Considerable common genes were assigned to pathways of sugar metabolism, mitogen-activated protein kinase, and circadian rhythm. The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses, hormone signaling, energy metabolism, starch metabolism, and circadian rhythm. These findings demonstrate the positive effect of auxin on AR induction, and uncovered the crucial roles of stress responses, hormone signaling and circadian rhythm in coordinating the physiological changes during the early phases of AR formation in mulberry stem hardwood cuttings.

CpSPL10-CpELF4 module involves in the negative regulation of flower bud differentiation in Chinese cherry.

SQUAMOSA promoter-binding protein-like (SPL) genes play a crucial role in regulating floral induction. Despite such importance, a comprehensive study of SPLs in Chinese cherry flower bud development has been absent. In this study, 32 CpSPL genes were identified. According to expression profiling, CpSPLs exhibited tissue-specific expression and distinct trends throughout flower bud differentiation. Specifically, CpSPL10 was greatly expressed at the beginning of the differentiation, and its role was further investigated. Its overexpression extended the vegetative growth of transgenic tobacco plants, delayed flowering by about 20 days. Moreover, the accumulation of NbELF4 (Early flowering 4) transcripts was enhanced due to the up-regulated levels of CpSPL10 in tobacco plants. ELF4 functions as a major element of the circadian clock; its high expression typically delays the transition from vegetative-to-reproductive growth. Further experiments revealed that CpSPL10 interacts with CpSPL9 or a transposase-derived transcription factor CpFRS5 (FAR1-RELATED SEQUENCE 5) and activates the expression of the downstream gene CpELF4. Notably, the GUS fusing reporter assay detected the activation of CpSPL10 and CpELF4 promoters in shoot apical meristems of transgenic Arabidopsis. These findings revealed the negative regulation of the CpSPL10-CpELF4 module in flower bud differentiation, providing references for supplementing the specific relationships among SPL, FRS, and ELF4.

Molecular characterization and evolutionary relationships of DOFs in four cherry species and functional analysis in sweet cherry.

The DOF (DNA binding with one finger) has multiple functions in plants. However, it has received little attention in the research field of cherries. In this study, the evolutionary relationship and molecular characterization of DOF in four cherry species were analyzed, revealing its expression pattern in sweet cherry. There are 23 members in Prunus avium cv. 'Tieton', 88 in Prunus cerasus, 53 in Cerasus × yedoensis, and 27 in Cerasus serrulata. Most of these genes are intron-less or non-intron, with a conserved C2-C2 domain. Due to heterozygosity and chromosomal ploidy, whole-genome duplication (WGD) events occur to varying degrees, and DOF genes are contracted during evolution. Furthermore, these genes are affected by purifying selection pressure. Under low-temperature treatment, the expression of PavDOF2 and PavDOF18 were significantly up-regulated, while that of PavDOF16 is significantly down-regulated. The expression of PavDOF9, PavDOF12, PavDOF14, PavDOF16, PavDOF17, PavDOF18, and PavDOF19 exhibits an increasing trend during flower development and varies during sweet cherry fruit development. PavDOF1, PavDOF8, PavDOF9, and PavDOF15 are localized in the nucleus but is not transcriptionally active. The findings systemically demonstrate the molecular characteristics of DOF in different cherry varieties, providing a basis for further research on the functions of these genes.

Genome-wide characterization of chalcone synthase genes in sweet cherry and functional characterization of CpCHS1 under drought stress.

Cherries are one of the important fruit trees. The growth of cherry is greatly affected by abiotic stresses such as drought, which hinders its development. Chalcone synthase (CHS, EC 2.3.1.74) is a crucial rate-limiting enzyme in the flavonoid biosynthetic pathway that plays an important role in regulating plant growth, development, and abiotic stress tolerance. In the current study, three genes encoding chalcone synthase were identified in the genome of sweet cherry (Prunus avium L.). The three genes contained fewer introns and showed high homology with CHS genes of other Rosaceae members. All members are predicted to localize in the cytoplasm. The conserved catalytic sites may be located at the Cys163, Phe214, His302, and Asn335 residues. These genes were differentially expressed during flower bud dormancy and fruit development. The total flavonoid content of Chinese cherry (Cerasus pseudocerasus Lindl.) was highest in the leaves and slightly higher in the pulp than in the peel. No significant difference in total flavonoid content was detected between aborted kernels and normally developing kernels. Overexpression of Chinese cherry CpCHS1 in tobacco improved the germination frequency of tobacco seeds under drought stress, and the fresh weight of transgenic seedlings under drought stress was higher than that of the wild type, and the contents of SOD, POD, CAT, and Pro in OE lines were significantly increased and higher than WT under drought stress. These results indicate cherry CHS genes are conserved and functionally diverse and will assist in elucidating the functions of flavonoid synthesis pathways in cherry and other Rosaceae species under drought stress.

Determinants of Shoot Biomass Production in Mulberry: Combined Selection with Leaf Morphological and Physiological Traits.

Physiological and morphological traits have a considerable impact on the biomass production of fast-growing trees. To compare cultivar difference in shoot biomass and investigate its relationships with leaf functional traits in mulberry, agronomic traits and 20 physiological and morphological attributes of 3-year-old mulberry trees from eight cultivars growing in a common garden were analyzed. The cultivars Xiang7920, Yu711, and Yunsang2 had higher shoot fresh biomass (SFB), which was closely associated with their rapid leaf expansion rate, large leaf area, and high stable carbon isotope composition (δ13C). Conversely, the cultivars 7307, Husang32, Wupu, Yunguo1, and Liaolu11 were less productive, and this was primarily the result of slower leaf expansion and smaller leaf size. Growth performance was negatively correlated with leaf δ13C and positively correlated with the total nitrogen concentration, indicating that a compromise exists in mulberry between water use efficiency (WUE) (low δ13C) and high nitrogen consumption for rapid growth. Several morphological traits, including the maximum leaf area (LAmax), leaf width and length, petiole width and length, leaf number per shoot, and final shoot height were correlated with SFB. The physiological traits that were also influential factors of shoot biomass were the leaf δ13C, the total nitrogen concentration, and the water content. Among the studied leaf traits, LAmax, leaf δ13C, and concentrations of chlorophyll a and b were identified as the most representative predictor variables for SFB, accounting for 73% of the variability in SFB. In conclusion, a combination of LAmax, leaf δ13C, and chlorophyll should be considered in selection programs for high-yield mulberry cultivars.