Genome-Wide Identification, Expression, and Interaction Analysis of BEL-Like Homeodomain Gene Family in Peach.

BEL1-like homeodomain (BLH) family genes as homeodomain transcription factors are found ubiquitously in plants to play important regulatory roles in reproductive development, morphological development, and stress response. Although BLH proteins have been reported in some species, there is little information about BLH genes in peach. In this study, we identified 11 peach PpBLH genes based on the conserved domain. Phylogenetic analysis suggested that the PpBLH proteins could be divided into five groups, which might be involved in different aspects of morphogenesis. Genomics structure analysis revealed that there were four exons in the PpBLH gene, and the length of the third exon was 61 bp. Chromosomal location analysis showed that the PpBLH genes were not distributed uniformly on six chromosomes. Promoter analysis showed that the promoter sequences of six PpBLH genes contained multiple cis-acting elements for hormones and stress. Six PpBLH genes were cloned by RT-PCR, and PpBLH1, PpBLH4, and PpBLH7 showed different expression patterns in the tested fruits under common temperature and high temperature. Y2H results indicated that PpBLH7 andPpBLH10 interacted with the PpOFP6 protein, and PpBLH1 interacted with the PpOFP1, PpOFP2, PpOFP4, and PpOFP13 proteins. These results provide new insight for further study of PpBLH genes, and construction of regulatory networks of PpBLH proteins in the growth, development, and stress response of peach.

The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants.

In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees.

[Genome-wide identification and expression analysis of the WRKY gene family in peach].

The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles.

KNOTTED1-like homeobox (KNOX) transcription factors - Hubs in a plethora of networks: A review.

KNOX (KNOTTED1-like HOMEOBOX) belongs to a class of important homeobox genes, which encode the homeodomain proteins binding to the specific element of target genes, and widely participate in plant development. Advancements in genetics and molecular biology research generate a large amount of information about KNOX genes in model and non-model plants, and their functions in different developmental backgrounds are gradually becoming clear. In this review, we summarize the known and presumed functions of the KNOX gene in plants, focusing on horticultural plants and crops. The classification and structural characteristics, expression characteristics and regulation, interacting protein factors, functions, and mechanisms of KNOX genes are systematically described. Further, the current research gaps and perspectives were discussed. These comprehensive data can provide a reference for the directional improvement of agronomic traits through KNOX gene regulation.

Molecular cloning and functional characterization of MdSOS2 reveals its involvement in salt tolerance in apple callus and Arabidopsis.

Plants respond to various environmental stresses by activating "stress genes". CIPKs (CBL-interacting protein kinases) family genes play an important role in the process of stress response. In this study, a CIPK gene MdSOS2 was isolated from apple (Malus × Domestica). Sequence alignment and phylogenetic analysis showed that it is highly similar with Arabidopsis AtSOS2 and contained the conserved domains and motifs. Expression analysis demonstrated that MdSOS2 expressed in all tested organs at different levels, and positively in response to salt stress. Furthermore, the ectopic expression of MdSOS2 complemented the function of Arabidopsis sos2 mutant, and conferred enhanced salt tolerance to the transgenic Arabidopsis. Yeast two-hybrid assay indicated that the N-terminal of MdSOS2 protein physically interacted with MdSOS3 and AtSOS3, respectively, suggesting that SOS pathway operates in apple tree. Finally, MdSOS2 overexpression enhanced, while its suppression reduced the tolerance to salt in transgenic apple calluses, indicating that MdSOS2 acts as a positive regulator in response to salt stress in apple.

Polycomb-group protein SlMSI1 represses the expression of fruit-ripening genes to prolong shelf life in tomato.

Polycomb-group (PcG) protein MULTICOPY SUPPRESSOR OF IRA1 (MSI1) protein is an evolutionarily conserved developmental suppressor and plays a crucial role in regulating epigenetic modulations. However, the potential role and function of MSI1 in fleshy fruits remain unknown. In this study, SlMSI1 was cloned and transformed into tomato to explore its function. The quantitative real-time PCR results showed that SlMSI1 was highly expressed in flowers and fruits and that its transcript and protein levels were significantly decreased in fruits after the breaker stage. Additionally, SlMSI1-overexpressing transgenic tomatoes displayed abnormal non-ripening fruit formation, whereas its suppression promoted fruit ripening in transgenic tomatoes. Quantitative real-time PCR assays also showed that RIN and its regulons were decreased in SlMSI1 overexpression transgenic tomato fruits. Furthermore, RNA-seq analysis demonstrated that SlMSI1 inhibits fruit ripening by negatively regulating a large set of fruit-ripening genes in addition to RIN and its regulons. Finally, genetic manipulation of SlMSI1 and RIN successfully prolonged the fruit shelf life by regulating the fruit-ripening genes in tomato. Our findings reveal a novel regulatory function of SlMSI1 in fruit ripening and provide a new regulator that may be useful for genetic engineering and modification of fruit shelf life.

MdVHA-A encodes an apple subunit A of vacuolar H(+)-ATPase and enhances drought tolerance in transgenic tobacco seedlings.

Vacuole H(+)-ATPases (VHAs) are plant proton pumps, which play a crucial role in plant growth and stress tolerance. In the present study, we demonstrated that the apple vacuolar H(+)-ATPase subunit A (MdVHA-A) is highly conserved with subunit A of VHA (VHA-A) proteins from other plant species. MdVHA-A was expressed in vegetative and reproductive organs. In apple in vitro shoot cultures, expression was induced by polyethylene glycol (PEG)-mediated osmotic stress. We further verified that over-expression of MdVHA-A conferred transgenic tobacco seedlings with enhanced vacuole H+-ATPase (VHA) activity and improved drought tolerance. The enhanced PEG-mimic drought response of transgenic tobacco seedlings was related to an extended lateral root system (dependent on auxin translocation) and more efficient osmotic adjustment. Our results indicate that MdVHA-A is a candidate gene for improving drought tolerance in plants.

Ectopic expression of an apple apomixis-related gene MhFIE induces co-suppression and results in abnormal vegetative and reproductive development in tomato.

It has been well documented that FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) plays important regulatory roles in diverse developmental processes in model plant Arabidopsis thaliana. However, it is largely unknown how FIE genes function in economically important crops. In this study, MhFIE gene, which was previously isolated from apomictic tea crabapple (Malus hupehensis Redh. var. pingyiensis), was introduced into tomato. The hemizygous transgenic tomato lines produced curly leaves and decreased in seed germination. In addition, the co-suppression of the transgenic MhFIE and endogenous (SlFIE) genes occurred in homozygous transgenic tomatoes. As a result, FIE silencing brought about abnormal phenotypes during reproductive development in tomato, such as increased sepal and petal numbers in flower, a fused ovule and pistil and parthenocarpic fruit formation. A yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) demonstrated that MhFIE interacted with a tomato protein, EZ2 (SlEZ2). Its ectopic expression and SlFIE co-suppression notably influenced the expression of genes associated with leaf, flower, and fruit development. Therefore, together with other PcG proteins, FIE was involved in the regulation of vegetative and reproductive development by modulating the expression of related genes in plants.

Functional characterization of an apple apomixis-related MhFIE gene in reproduction development.

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple.

MdVHP1 encodes an apple vacuolar H(+)-PPase and enhances stress tolerance in transgenic apple callus and tomato.

Vacuolar H(+)-translocating inorganic pyrophosphatase (VHP, EC 3.6.1.1) is an electrogenic proton pump, which is related to growth as well as abiotic stress tolerance in plants. In this study, a VHP gene MdVHP1 was isolated from apple. The alignment of nucleotide and amino acid sequences showed that it encoded a type I VHP protein. It expressed in vegetative and reproductive organs, and its expression was induced by salt, PEG-mediated osmotic stress, cold and heat in apple in vitro shoot cultures. MdVHP1 expression showed a similar pattern in different apple tissues, but different change dynamics in response to abiotic stresses, compared with MdVHP2 (another MdVHP gene in apple). MdVHP1 overexpression enhanced tolerance to salt, PEG-mimic drought, cold and heat in transgenic apple calluses, which was related to an increased accumulation of proline and decreased MDA content compared with control calluses. In addition, MdVHP1 overexpression confers improved tolerance to salt and drought in transgenic tomato, along with an increased ion accumulation, high RWC and low solute potential compared with wild type. These results indicate that MdVHP1 is an important regulator for plant tolerance to abiotic stresses by modulating internal stores of ions and solutes.

Expression analysis and functional characterization of apple MdVHP1 gene reveals its involvement in Na(+), malate and soluble sugar accumulation.

The vacuolar H(+)-pyrophosphatase (VHP) is a proton pump, which energizes transport across the tonoplast. The contributions of VHP to ion, organic acid and sugar storage are unclear in fruit. Here we characterized the role of an apple vacuolar H(+)-pyrophosphatase gene (MdVHP1) in Na(+), malate and soluble sugar accumulation. MdVHP1 expression was consistent with VHP activities in apple fruits at most developmental stages, and highly significantly correlated with Na(+) concentration during fruit development. In apple fruits treated in vitro (i.e., fruit-bearing branches were cultured in Hoagland solution containing each of salt, malate and sucrose, respectively, or irradiated by blue light), MdVHP1 expression was significantly correlated with Na(+) and malate transporter genes MdNHX1 and MdtDT, while subunit A of the vacuolar H(+)-ATPase (MdVHA-A) was significantly correlated with MdtDT and sucrose transporter gene (MdSUT1). In addition, MdVHP1 overexpression noticeably promoted Na(+) and malate accumulation, but slightly increased soluble sugar accumulation in transgenic apple callus and tomato fruit, partially by regulating transporter genes MdNHX1, MdtDT and MdSUT1. Taken together, it appears that MdVHP1 favorably contributes to Na(+), malate and soluble sugar accumulation in apple fruit.

The functions of an apple cytosolic malate dehydrogenase gene in growth and tolerance to cold and salt stresses.

It is well-known that cytosolic NAD-dependent malate dehydrogenase (cyMDH; l-malate:NAD-oxidoreductase; EC 1.1.1.37) is an enzyme crucial for malic acid synthesis in the cytosol. Nothing is known about cyMDH in growth and stress tolerance. Here we characterised the role of the apple cyMDH gene (MdcyMDH, GenBank ID: DQ221207) in growth and tolerance to cold and salt stresses. MdcyMDH transcripts were highly accumulated in vigorously growing apple tissues, organs and suspension cells. In addition, MdcyMDH was sensitive to cold and salt stresses. MdcyMDH overexpression favourably contributed to cell and plant growth and conferred stress tolerance both in the apple callus and tomato. Taken together, our results indicated that MdcyMDH is involved in plant and cell growth as well as the tolerance to cold and salt stresses.

[Estimation of the position of right-sided double-lumen endobronchial tubes with spirometry in elderly patients].

OBJECTIVE: To study the accuracy of position estimation of right-sided double-lumen endobronchial tubes (DLTs) without carinal hook in elderly patients according to the changes of inspiratory peak airway pressure (Ppeak), lung compliance (Cdyn), pressure-volume loop (P-V loop), and flow-volume loop (F-V loop). METHODS: Ninety-six elderly patients undergoing thoracic surgery were intubated with right-sided Mallinckrodt DLTs, the depth of which was determined based on the regressive equation. After 15 min of two lung ventilation (TLV), the Ppeak showed a more than 50% increase from the baseline in 22 patients when switching to one lung ventilation (OLV) with Cdyn less than 50% of the baseline and Ppeak in excess of 22 cmH(2)O. In these 22 cases, the position of the DLT was determined by fiberoptic bronchoscope (FOB) and adjusted to the precise position at 15 min of OLV. RESULTS: DLTs were inserted into the right middle bronchi in 19 cases and the right lower lobe bronchi in 3 cases. At 124-/+39 s after OLV, the SpO(2) began to decrease, and the Ppeak of OLV increased by 91.0% and Cdyn decreased by 62.7%. The slope of P-V curve was reduced and the P-V loop extended, with reduced increment of the expiratory limb and the area of the F-V loop, so that the tips of the DLT were withdrawn by 0.5 to 1.9 cm. The Ppeak of OLV increased only by 43.4% when DLT was in correct position, and was Cdyn decreased by 33.6% ( P<0.01). CONCLUSION: When changes of Ppeak and Cdyn in excess of above guidelines occurs after switching from TLV to OLV in the elderly patients, FOB should be applied to determine the tip position of DLT before SpO(2) reduction takes place.