The RALF1-FERONIA interaction modulates endocytosis to mediate control of root growth in Arabidopsis.

The interaction between the receptor-like kinase (RLK) FERONIA (FER) and the secreted peptide RAPID ALKALINIZATION FACTOR1 (RALF1) is vital for development and stress responses in Arabidopsis Ligand-induced membrane dynamics affect the function of several RLKs, but the effects of the RALF1-FER interaction on the dynamics of FER and the ensuing effects on its functionality are poorly understood. Here, we show that RALF1 modulated the dynamics and partitioning of FER-GFP at the plasma membrane (PM). Moreover, FER was internalized by both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) under steady-state conditions. After RALF1 treatment, FER-GFP internalization was primarily enhanced via the CME pathway, raising FER-GFP levels in the vacuole. RALF1 treatment also modulated trafficking of other PM proteins, such as PIN2-GFP and BRI1-GFP, increasing their vacuolar levels by enhancing their internalization. Importantly, blocking CME attenuated RALF1-mediated root growth inhibition independently of RALF1-induced early signaling, suggesting that the RALF1 can also exert its effects via the CME pathway. These findings reveal that the RALF1-FER interaction modulates plant growth and development, and this might also involve endocytosis of PM proteins.

Genome-wide identification of the NRAMP gene family in Populus trichocarpa and their function as heavy metal transporters.

The natural resistance-associated macrophage protein (NRAMP) gene family plays a key role in essential mineral nutrient homeostasis, as well as toxic metal accumulation, translocation, and detoxification. Although the NRAMP family genes have been widely identified in various species, they still require to be analyzed comprehensively in tree species. In this study, a total of 11 NRAMP members (PtNRAMP1-11) were identified in Populus trichocarpa, a woody model plant, and further subdivided into three groups based on phylogenetic analysis. Chromosomal location analysis indicated that the PtNRAMP genes were unevenly distributed on six of the 19 Populus chromosomes. Gene expression analysis indicated that the PtNRAMP genes were differentially responsive to metal stress, including iron (Fe) and manganese (Mn) deficiency, as well as Fe, Mn, zinc (Zn), and cadmium (Cd) toxicity. Furthermore, the PtNRAMP gene functions were characterized using a heterologous yeast expression system. The results showed that PtNRAMP1, PtNRAMP2, PtNRAMP4, PtNRAMP9, PtNRAMP10, and PtNRAMP11 displayed the ability to transport Cd into yeast cells. In addition, PtNRAMP1, PtNRAMP6, and PtNRAMP7 complemented the Mn uptake mutant, while PtNRAMP1, PtNRAMP6, PtNRAMP7, and PtNRAMP9 complemented the Fe uptake mutant. In conclusion, our findings revealed the respective functions of PtNRAMPs during metal transport as well as their potential role in micronutrient biofortification and phytoremediation.

The VAMP-associated protein VAP27-1 plays a crucial role in plant resistance to ER stress by modulating ER-PM contact architecture in Arabidopsis.

The endoplasmic reticulum (ER) and the plasma membrane (PM) form ER-PM contact sites (EPCSs) that allow the ER and PM to exchange materials and information. Stress-induced disruption of protein folding triggers ER stress, and the cell initiates the unfolded protein response (UPR) to resist the stress. However, whether EPCSs play a role in ER stress in plants remains unclear. VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP)-ASSOCIATED PROTEIN 27-1 (VAP27-1) functions in EPCS tethering and is encoded by a family of 10 genes (VAP27-1-10) in Arabidopsis thaliana. Here, we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4 (vap27-1/3/4) triple mutant lacking three of the key VAP27 family members in Arabidopsis. The vap27-1/3/4 mutant exhibits defects in ER-PM connectivity and EPCS architecture, as well as excessive UPR signaling. We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress. Moreover, the spatiotemporal dynamics of VAP27-1 at the PM increase ER-PM connectivity and enhance Arabidopsis resistance to ER stress. In addition, we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling. Taken together, these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants, providing additional clues for improving plant broad-spectrum resistance to different stresses.

Genome-wide identification of PLATZ genes related to cadmium tolerance in Populus trichocarpa and characterization of the role of PtPLATZ3 in phytoremediation of cadmium.

Plant AT-rich sequence and zinc-binding (PLATZ) proteins are a class of plant-specific zinc finger transcription factors that perform critical functions in plant development and resistance. However, the function of PLATZs in heavy metal tolerance has not yet been investigated. Moreover, only a few PLATZ proteins have been functionally characterized in tree species. In this study, we identified 18 PtPLATZ genes in Populus trichocarpa, an important woody model plant, and classified them into five groups. PtPLATZ genes attributed to the same clade usually possess similar exon-intron structures containing two or three introns, as well as a similar motif composition. Furthermore, chromosomal location analysis indicated an uneven distribution of PtPLATZ genes on 13 of the 19 Populus chromosomes. Promoter cis-acting element prediction and gene expression analysis showed that PtPLATZ genes were highly responsive to heavy metal stress. Heterologous yeast expression revealed that PtPLATZ1, PtPLATZ2, PtPLATZ3, PtPLATZ4, PtPLATZ8 and PtPLATZ9 are significantly involved in Cd tolerance. In addition, transgenic expression of PtPLATZ3 significantly enhanced Cd tolerance and accumulation, slowed the decline in chlorophyll content, maintained membrane integrity in Populus, and increased the expression of genes related to Cd tolerance and accumulation. In conclusion, our results suggest the potential of PtPLATZ3 to improve Cd tolerance and accumulation in Populus, which is of great significance for phytoremediation.

Comprehensive analysis of lncRNA-mRNA regulatory network in Populus associated with xylem development.

Long noncoding RNAs (lncRNAs) play essential roles in numerous biological processes in plants, such as regulating the gene expression. However, only a few studies have looked into their potential functions in xylem development. High-throughput sequencing of P. euramericana 'Zhonglin46' developing and mature xylem was performed in this study. Through sequencing analysis, 14,028 putative lncRNA transcripts were identified, including 4525 differentially expressed lncRNAs (DELs). Additional research revealed that in mature xylem, a total of 2320 DELs were upregulated and 2205 were downregulated compared to developing xylem. Meanwhile, there were a total of 8122 differentially expressed mRNAs (DEMs) that were upregulated and 16,424 that were downregulated in mature xylem compared with developing xylem. The cis- and trans-target genes of DELs were analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, which indicated that these DELs participate in controlling the phenylpropanoid and lignin biosynthesis pathway as well as the starch and sucrose metabolism pathway. Among the cis-regulated DELs, LNC_006291, LNC_006292, and LNC_006532 all participate in regulating multiple HCT gene family membranes. As targets, POPTR_001G045900v3 (CCR2) and POPTR_018G063500v3 (SUS) both have only one cis-regulatory lncRNA, referred to as LNC_000057 and LNC_006212, respectively. Moreover, LNC_004484 and two DELs named LNC_008014 and LNC_010781 were revealed to be important nodes in the co-expression network of trans-lncRNAs and mRNAs associated to the lignin biosynthesis pathway and cellulose and xylan biosynthetic pathways, respectively. Finally, quantitative real-time PCR (qRT-PCR) was used to confirme 34 pairs of lncRNA-mRNA. Taken together, these findings may help to clarify the regulatory role that lncRNAs play in xylem development and wood formation.

Insights from multi-omics integration into seed germination of Taxus chinensis var mairei.

The transition from deep dormancy to seed germination is essential for the life cycle of plants, but how this process occurs in the gymnosperm Chinese yew (Taxus chinensis var mairei), the natural source of the anticancer drug paclitaxel, remains unclear. Herein, we analyse the transcriptome, proteome, spatial metabolome, and spatial lipidome of the Chinese yew and present the multi-omics profiles of dormant and germinating seeds. Our results show that abscisic acid and gibberellic acid 12 homoeostasis is closely associated with gene transcription and protein translation, and the balance between these phytohormones thereby determines if seeds remain dormant or germinate. We find that an energy supply of carbohydrates from glycolysis and the TCA cycle feed into the pentose phosphate pathway during seed germination, and energy supplied from lipids are mainly derived from the lipolysis of triacylglycerols. Using mass spectrometry imaging, we demonstrate that the spatial distribution of plant hormones and phospholipids has a remarkable influence on embryo development. We also provide an atlas of the spatial distribution of paclitaxel C in Chinese yew seeds for the first time. The data from this study enable exploration of the germination mechanism of Chinese yew seeds across several omics levels.

Codon usage bias predicts the functional MYB10 gene in Populus.

Analysis of codon usage bias (CUB) in different species can reveal the patterns of genetic information transfer across those species. To better understand the characteristics of MYB10-a key regulator of anthocyanin biosynthesis-and identify the true (functional) MYB10 gene among the two candidates in Populus, we analysed the coding sequences of MYB10 genes in 10 different species using Codon W, CHIPS, CUSP, and CAI. Majority of the optimal amino acid codons of MYB10 genes ended with A/U, and GGA, UCA, GCA, AGA, and CCA were over-represented in all plant species studied. Among the two most promising MYB10 gene candidates in Populus, Potri.17G125700 shared a higher similarity of codon usage with MYB10 genes from other plant species, suggesting that it encodes the functional MYB10 in Populus. We verified this speculation by cloning both candidate MYB10 genes from Populus into vectors to produce transiently transformed seedlings. Colour phenotypes and anthocyanin content of the transiently transformed seedlings indicated that Potri.17G125700 encodes the true MYB10 transcription factor, which positively regulates anthocyanin accumulation in Populus. Furthermore, CUB analysis was used to select the most promising MYB12 candidate in Malus sp. (crabapple). Our results demonstrate the effectiveness of CUB analysis as a promising method to identify the functional gene from a set of candidates in long-living plants with complex genetics.

Factor Analysis of MYB Gene Expression and Flavonoid Affecting Petal Color in Three Crabapple Cultivars.

Flavonoid biosynthesis has received much attention concerning the structural genes and expression of the associated transcription factors (TFs). In the present study, we examined the gene expression patterns for petals of three colors using a statistical method. Factor analysis was successfully used to examine the expression patterns most present during regulation. The first expression patterns in the white and red petals were clearly demonstrated and have revealed different mechanisms of producing the proper components, whereas that in the pink petals was more complex, requiring factor analysis to supplement the other results. Combining the results of the correlation analysis between TFs and structural genes, the effects of each TF on the main expression pattern in each cultivar were determined. Moreover, McMYB10 was implicated in the regulation of the gene expression pattern in red petals, and McMYB5 was implicated in the maintenance of the balance of the pigment components and proanthocyanin (PA) production in cooperation with McMYB4 to generate pigmentation in the pink petals.

Flavonoid Accumulation Plays an Important Role in the Rust Resistance of Malus Plant Leaves.

Cedar-apple rust (Gymnosporangium yamadai Miyabe) is a fungal disease that causes substantial injury to apple trees and results in fruit with reduced size and quality and a lower commercial value. The molecular mechanisms underlying the primary and secondary metabolic effects of rust spots on the leaves of Malus apple cultivars are poorly understood. Using HPLC, we found that the contents of flavonoid compounds, especially anthocyanin and catechin, were significantly increased in rust-infected symptomatic tissue (RIT). The expression levels of structural genes and MYB transcription factors related to flavonoid biosynthesis were one- to seven-fold higher in the RIT. Among these genes, CHS, DFR, ANS, FLS and MYB10 showed more than a 10-fold increase, suggesting that these genes were expressed at significantly higher levels in the RIT. Hormone concentration assays showed that the levels of abscisic acid (ABA), ethylene (ETH), jasmonate (JA) and salicylic acid (SA) were higher in the RIT and were consistent with the expression levels of McNCED, McACS, McLOX and McNPR1, respectively. Our study explored the complicated crosstalk of the signal transduction pathways of ABA, ETH, JA and SA; the primary metabolism of glucose, sucrose, fructose and sorbitol; and the secondary metabolism of flavonoids involved in the rust resistance of Malus crabapple leaves.

MYBs affect the variation in the ratio of anthocyanin and flavanol in fruit peel and flesh in response to shade.

Fruit pigment accumulation, which represents an important indicator of nutrient quality and appearance value, is often affected by low light under rain, cloud, fog and haze conditions during the veraison period. It is not known whether continuous low light interferes with the production and accumulation of secondary metabolites in veraison fruit. In this paper, we measured pigments and the transcriptional level of genes related to secondary metabolites, i.e., flavonoid biosynthesis in the peel and flesh of Malus crabapple 'Radiant' fruit in response to normal light and shade from 10th July to 30th August. The results showed crosstalk between the flavonoid biosynthetic genes and the involvement of key transcription factors such as McMYB4, McMYB7, McMYB10, and McMYB16 in the regulation of the ratio of anthocyanins and flavanols, which accounted for the different colouration of the fruit peel and flesh under shade conditions. A model is proposed for the regulation of the flavonoid pathway in the peel and flesh of 'Radiant' fruit based on our study results. Moreover, the molecular mechanism for 'Radiant' fruit colouration provides reference information for understanding the light regulatory mechanism involved in the biosynthesis of flavonoids and for designing the next generation of apple breeding.

Changes of interleukin-6 and related factors as well as gastric intramucosal pH during colorectal and orthopaedic surgical procedures.

OBJECTIVE: To investigate the changes of perioperative serum levels of interleukin-6 (IL-6), C-reactive protein (CRP), and cortisol, as well as gastric intramucosal pH (pHi) and plasma lactate, aiming to compare systemic changes and tissue perfusion during colorectal and orthopaedic surgical procedures. METHODS: Twenty patients were randomly assigned to two groups, 10 cases of operation on vertebral canal, 10 cases of colorectal radical operation. Venous blood was drawn at 1 day before operation, 2, 4, and 6 hours following skin incision, and 1 day after operation, in order to measure serum IL-6, CRP, and cortisol. pHi and plasma lactate were also measured at the same time points. RESULTS: Serum concentrations of IL-6 and cortisol increased gradually following operation, reaching the peak value at 6 hours from the beginning of operation. CRP was not detectable until the first day after operation. Peak concentration of IL-6 had positive relationship with CRP. These variables changed more significantly in colorectal group than that in orthopaedic group (P < 0.05). pHi decreased gradually, reaching the lowest level at 4 hours from the beginning of operation, and to more extent in colorectal group than that in orthopaedic group (P < 0.05). CONCLUSION: IL-6 may reflect tissue damage more sensitively than CRP. Colorectal surgery might induce systemic disorder to more extent, in terms of immuno-endocrinal aspect as well as tissue perfusion, reflected with pHi.

An Apple Fruit Fermentation (AFF) Treatment Improves the Composition of the Rhizosphere Microbial Community and Growth of Strawberry (Fragaria × ananassa Duch 'Benihoppe') Seedlings.

Plant growth can be promoted by the application of apple fruit fermentation (AFF), despite unclear of the underlying mechanisms, the effects involved in AFF on rhizosphere microorganisms have been hypothesized. We investigated the consequences of applying AFF alone or in combination with Bacillus licheniformis to strawberry tissue culture seedlings in vitro, the analyses of Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA were performed to determine AFF effects on rhizosphere. Moreover, the growth index and antioxidant enzyme activities were determined 30 days after treatments. We identified five dominant bacteria in AFF: Coprinus atramentarius, Bacillus megaterium, Bacillus licheniformis, Weissella and B. subtilis. The greatest number of bacterial species were observed in the rhizosphere of control matrix (water treated), and the lowest diversity appeared in the rhizosphere soil treated with 108 cfu/mL B. licheniformis alone. Combining AFF plus B. licheniformis in one treatment resulted in the largest leaf area, plant height, root length, plant weight, and the markedly higher activities of antioxidant enzymes. We conclude that a combination of AFF plus B. licheniformis treatment to matrix can increase antioxidant enzymes activities in strawberry seedlings, optimize the status of rhizosphere microbial, and promote plant growth.

[Orexin A and neuropeptide Y levels in plasma and hypothalamus of rats with chronic renal failure].

OBJECTIVE: To explore the changes of orexin A and neuropeptide (NPY) in plasma and hypothalamus of rats with chronic renal failure (CRF). METHODS: 41 male Wister rats weighing 200 approximately 250 g were randomly divided into three groups: normal group, sham operation group, and CRF group (with the right kidney and 2/3 of the left kidney resected). A certain number of rats were decapitated 4, 8,and 12 weeks after respectively. Their hypothalami were removed and blood collected. Radioimmunoassay was used to measure the levels of orexin A and NPY in hypothalamus and plasma. Automatic biochemical analyzer was used to measure the serum creatinine. RESULTS: The serum creatinine level of CRF rats was both significantly higher than those of the sham operation rats at week 8 and week 12, respectively. The plasma orexin A level of CRF rats at week 12 was 264 pg/ml +/- 62 pg/ml, significantly higher than that of sham operation group (183 pg/ml +/- 56pg/ml, P = 0.039). The hypothalamus orexin A level of CRF rats were 10.5 fmol/mg +/- 2.7 fmol/mg wet weight at week 12, significantly lower than that of sham operation rats (17.4 fmol/mg +/- 3.9 fmol/mg wet weight, P = 0.023). The plasma NPY levels of CRF rats at week 8 and week 12 were significantly higher than those of the sham operation rats (7.1 pmol/ml +/- 1.7 pmol/ml vs 5.0 pmol/ml +/- 0.5 pmol/ml, P = 0.01; and 7.9 pmol/ml +/- 1.1 pmol/ml vs 4.8 pmol/ml +/- 1.1 pmol/ml, P = 0.0008). The hypothalamus NPY level of CRF rats at week 12 were 70 fmol/mg +/- 23 fmol/mg wet weight, significantly lower than that of the sham operation rats (113 fmol/mg +/- 31 fmol/mg wet weight, P = 0.03). CONCLUSION: Loss of renal function may diminish the excretion of orexin A and neuropeptide. The lowering of hypothalamus orexin A and neuropeptide Y levels may be one of the causes inducing anorexia in CRF.