A COBRA family protein, PtrCOB3, contributes to gelatinous layer formation of tension wood fibers in poplar.

Angiosperm trees usually develop tension wood (TW) in response to gravitational stimulation. TW comprises abundant gelatinous (G-) fibers with thick G-layers primarily composed of crystalline cellulose. Understanding of the pivotal factors governing G-layer formation in TW fiber remains elusive. This study elucidates the role of a Populus trichocarpa COBRA family protein, PtrCOB3, in the G-layer formation of TW fibers. PtrCOB3 expression was upregulated, and its promoter activity was enhanced during TW formation. Comparative analysis with wild-type trees revealed that ptrcob3 mutants, mediated by Cas9/gRNA gene editing, were incapable of producing G-layers within TW fibers and showed severely impaired stem lift. Fluorescence immunolabelling data revealed a dearth of crystalline cellulose in the tertiary cell wall (TCW) of ptrcob3 TW fibers. The role of PtrCOB3 in G-layer formation is contingent upon its native promoter, as evidenced by the comparative phenotypic assessments of pCOB11::PtrCOB3, pCOB3::PtrCOB3, and pCOB3::PtrCOB11 transgenic lines in the ptrcob3 background. Overexpression of PtrCOB3 under the control of its native promoter expedited G-layer formation within TW fibers. We further identified three transcription factors that bind to the PtrCOB3 promoter and positively regulate its transcriptional levels. Alongside the primary TCW synthesis genes, these findings enable the construction of a two-layer transcriptional regulatory network for the G-layer formation of TW fibers. Overall, this study uncovers mechanistic insight into TW formation, whereby a specific COB protein executes the deposition of cellulose, and consequently, G-layer formation within TW fibers.

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth.

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.

Transcriptomic landscape of staminate catkins development during overwintering process in Betula platyphylla.

Betula platyphylla, belonging to the cold-specialized lineage Betulaceae, exhibits a unique reproductive strategy where staminate catkins emerge in the first summer and undergo an overwintering process, culminating in flowering in the following year. However, the underlying regulatory mechanism remains unclear. In this study, we investigated the male germline development of B. platyphylla in four distinct stages: microsporocytes in Oct. (S1), uninuclear microspores from Dec. (S2) to Mar. of the following year (S3), and bicellular microspores in Apr. (S4). We performed RNA sequencing on mature pollen and the four stages of staminate catkins. Using weighted gene co-expression network analysis (WGCNA), we identified five highly correlated gene modules with distinct expression profiles. These modules exhibited strong correlations with sugar metabolism, cell cycle, flowering, and cell wall dynamics, highlighting their dynamic roles during male germline developmental stages. During the overwintering process, we observed that the expression of transcription factors such as BpDUO1 and BpAMS at the appropriate developmental stages, suggests their significant roles in male germline development. The expression patterns of BpFLC and BpFT suggest their potential involvement in temperature perception during male reproductive development. These findings offer valuable insights into the reproductive success of plants adapting to cold environments.

OsSAP6 Positively Regulates Soda Saline-Alkaline Stress Tolerance in Rice.

BACKGROUND: Soil salinization is a worldwide environmental problem, especially in the arid and semiarid regions of northeastern China, which are heavily affected by soda saline-alkaline stress. At present, there is an urgent need to improve the soda saline-alkaline stress tolerance of rice. RESULTS: Stress-associated proteins are involved in regulating the abiotic stresses in plants. There are 18 members of the rice stress-associated protein (OsSAP) gene family. In this study, the expression levels of OsSAP6 in leaves and roots were upregulated with increasing NaHCO3 stress duration. OsSAP6 was located in nucleus and cytoplasm. The bud length and total root length of OsSAP6 overexpression rice were significantly longer than those of Lj11 (Oryza sativa longjing11) during germination stage, and the survival rates, plant height and malondialdehyde content at the seedling stage showed tolerance growth of saline-alkaline stress. The expression of OsCu/Zn-SOD, OsAPX2, and OsCAT1 in transgenic lines was increased significantly under SAE (soda saline-alkali soil eluent) stress. OsSAP6 interacts with OsPK5 according to yeast two-hybrid screening and luciferase complementation experiments. The expression of OsPK5 increased under NaHCO3 and H2O2 stress, and the overexpression of OsPK5 in rice improved soda saline-alkaline tolerance. CONCLUSION: Overexpression of OsSAP6 in rice significantly enhanced saline-alkaline tolerance compared with the wild type. It is speculated that OsSAP6 responds to soda salinity stress and interacts with OsPK5 to positively regulate soda saline-alkaline tolerance through ROS homeostasis. This study revealed the features of OsSAP6 involved in response to soda saline-alkaline stress and the interaction with OsPK5, which provided resources for breeding aimed at improving the soda saline-alkaline stress tolerance of rice.

Fatty Acid Biosynthesis Pathways Are Downregulated during Stigma Development and Are Critical during Self-Incompatible Responses in Ornamental Kale.

In Brassicaceae, the papillary cells of the stigma are the primary site of the self-incompatibility (SI) responses. SI preserves the genetic diversity by selectively rejecting irrelevant or incompatible pollen, thus promoting cross fertilization and species fitness. Mechanisms that regulate SI responses in Brassica have been studied mainly on the mature stigma that often undermines how stigma papillary cells attain the state of SI during development. To understand this, we integrated PacBio SMRT-seq with Illumina RNA-seq to construct a de novo full-length transcriptomic database for different stages of stigma development in ornamental kale. A total of 48,800 non-redundant transcripts, 31,269 novel transcripts, 24,015 genes, 13,390 alternative splicing, 22,389 simple sequence repeats, 21,816 complete ORF sequences, and 4591 lncRNAs were identified and analyzed using PacBio SMRT-seq. The Illumina RNA-seq revealed 15,712 differentially expressed genes (DEGs) and 8619 transcription factors. The KEGG enrichment analysis of 4038 DEGs in the "incompatibility" group revealed that the flavonoid and fatty acid biosynthesis pathways were significantly enriched. The cluster and qRT-PCR analysis indicated that 11 and 14 candidate genes for the flavonoid and fatty acid biosynthesis pathways have the lowest expression levels at stigma maturation, respectively. To understand the physiological relevance of the downregulation of fatty acid biosynthesis pathways, we performed inhibitor feeding assays on the mature stigma. The compatible pollination response was drastically reduced when mature stigmas were pre-treated with a fatty acid synthase inhibitor. This finding suggested that fatty acid accumulation in the stigmas may be essential for compatible pollination and its downregulation during maturity must have evolved as a support module to discourage the mounting of self-incompatible pollen.

Drought resistance of tobacco overexpressing the AfNAC1 gene of Amorpha fruticosa Linn.

Plants are often adversely affected by abiotic stresses such as drought, low temperature, and salinity during growth, and plant NAC-like transcription factors are involved in regulating growth and developmental processes in response to stresses such as drought and salinity. In this study, to investigate the function of AfNAC1, a co-expression network of AfNAC1 genes was constructed using gene expression data from the Chinese legume deciduous shrub, Amorpha fruticosa Linn. A 576 bp NAC transcription factor (AfNAC1 gene, MN180266) encoding 191 amino acids was isolated from Amorpha fruticosa seedlings by RT-PCR. qRT-PCR showed that the AfNAC1 gene was expressed in the roots, stems, leaves, and flowers of Amorpha fruticosa. However, drought stress significantly increased root expression, and the AfNAC1 protein was localized in the nucleus by green fluorescence detection. This study analyzed the drought resistance of overexpressing tobacco in depth. Under natural drought stress, the chlorophyll and antioxidant enzyme activities of overexpressing plants were significantly higher than those of wild-type (WT) plants, but the MDA content was lower than that of WT; after rehydration the Fv/Fm values of AfNAC1-overexpressing tobacco recovered faster than those of wild-type tobacco and rapidly reached the control levels; AfNAC1 may be involved in the regulation of the photosystem and indirectly in the regulation of the plant in response to drought stress.

Isolation and characterization of a J domain protein that interacts with ARC1 from ornamental kale (Brassica oleracea var. acephala).

KEY MESSAGE: A novel J domain protein, JDP1, was isolated from ornamental kale. The C-terminus of JDP1 specifically interacted with ARC1, which has a conserved role in self-incompatibility signaling. Armadillo (ARM)-repeat containing 1 (ARC1) plays a conserved role in self-incompatibility signaling across the Brassicaceae and functions downstream of the S-locus receptor kinase. Here, we identified a J domain protein 1 (JDP1) that interacts with ARC1 using a yeast two-hybrid screen against a stigma cDNA library from ornamental kale (Brassica oleracea var. acephala). JDP1, a 38.4-kDa protein with 344 amino acids, is a member of the Hsp40 family. Fragment JDP1(57-344), originally isolated from a yeast two-hybrid cDNA library, interacted specifically with ARC1 in yeast two-hybrid assays. The N-terminus of JDP1 (JDP1(1-68)) contains a J domain, and the C-terminus of JDP1 (JDP1(69-344)) contains an X domain of unknown function. However, JDP1(69-344) was required and sufficient for interaction with ARC1 in yeast two-hybrid assays and in vitro binding assays. Moreover, JDP1(69-344) regulated the trafficking of ARC1 from the cytoplasm to the plasma membrane by interacting with ARC1 in Arabidopsis mesophyll protoplasts. Finally, Tyr(8) in the JDP1 N-terminal region was identified to be the specific site for regulating the interaction between JDP1 and BoARC1 in yeast two-hybrid assays. Possible roles of JDP1 as an interactor with ARC1 in Brassica are discussed.

[Preparation and characterization of monoclonal antibodies against human CA50 and establishment of chemiluminescence immunoassay system].

OBJECTIVE: To prepare the anti-human carbohydrate antigen 50(CA50) monoclonal antibody (mAb), characterize its immunological features and establish a chemiluminescence immunoassay system with it. METHODS: BALB/c mice were immunized with human CA50 antigen for preparing mAb using hybridoma technique. Stable anti-CA50-secreting hybridoma cell lines were obtained after screening. The mAbs were purified using protein A after expanding culture. The chemiluminescence immunoassay system was established and evaluated in its linear range, accuracy, sensitivity, reproducibility, and the blood samples were tested with it. RESULTS: Four hybridoma cell lines were obtained respectively and their titers were above 1:10(8). Anti-CA50 mAbs nearly had no cross-reaction with CA125, CA153, CA199 and CA724. The linear detection of the chemiluminescence immunoassay system covered a range 0-500 U/mL. The recovery rate for accuracy was 107.08% and its sensitivity was 0.83 U/mL. The assay was highly repeatable [coefficient of variation (CV)<10%]. The correlation coefficient with the test of reference reagent was 0.96. CONCLUSION: The monoclonal antibodies against human CA50 had been screened and a chemiluminescence immunoassay system for human CA50 detection had been established successfully.

[Preparation and characterization of the monoclonal antibodies against human CA19-9 and establishment of a sandwich CLIA system].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 19-9 (CA19-9) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) detection system for CA19-9 in the human serum. METHODS: BALB/c mice were immunized with human CA19-9 antigen. The mAbs were obtained by hybridoma technique. The purity, titer, specificity and pairing of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity and repetitiveness after optimization of coating buffer, coating concentration and pipetting mode, and the serum sample was tested with it. RESULTS: Four mAbs named #1-1, #2-1, #3-1 and #4-1 were obtained against human CA19-9. The titers of the anti-CA19-9 mAbs were above 10(-8). The mAbs had nearly no cross-reaction with CA125, CA15-3 and CA72-4. The double-antibody sandwich CLIA system was established by #3-1 mAb and #2-1 mAb-HRP. After optimization, its property was detection range of 0-1 000 U/mL, limit detection of 2.0 U/mL, linear correlation coefficient of 0.9999. The results which were contrasted with Roche test showed that: Kappa>0.75, r(correlation coefficient)>0.9. CONCLUSION: The mAbs against human CA9-9 have been prepared and a sandwich CLIA system for detecting CA19-9 in the human serum has been established successfully.

[Preparation and characterization of monoclonal antibodies against human CA125 and establishment of a chemiluminescence immunoassay system].

OBJECTIVE: To prepare the anti-human carbohydrate antigen 125 (CA125) monoclonal antibody (mAb), and establish a chemiluminescence immunoassay detection system. METHODS: The BALB/c mice were immunized with human CA125 antigen. The hybridoma cells were obtained by cell fusion and screening. The mAbs were purified using protein A. The specificity, purity, isotype, and affinity of the mAbs were characterized and the sandwich ELISA system was established. The chemiluminescence immunoassay system was established and evaluated after optimization and selection of coating buffer, coating concentration, pipetting mode respectively, and the blood samples were tested with it. RESULTS: Three hybridoma cell lines named 30-1-4, 31-1-5 and 43-1-6 were obtained respectively. Anti-CA125 mAbs nearly had no cross-reaction with CA199, CA50 and CA724. The titres were above 1:10(8);. The chemiluminescence immunoassay system was established using 30-1-4 and 31-1-5. After optimization, the linear detection of the system covered a range 0-1000 U/mL, and its lowest detectable limit was 0.72 U/mL. The correlation coefficient with the results of the Roche test was above 0.90. CONCLUSION: With the mAbs against human CA125 we prepared, a chemiluminescence immunoassay system for human CA125 detection has been established successfully, which provides a basis for human CA125 detection and ovarian cancer diagnosis.

[Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection].

OBJECTIVE: To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection. METHODS: The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA (HTS-ELISA). Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established. RESULTS: The titers of mouse anti-sera reached 1:10(5);. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity (over 10(9); L/mol). Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg "d, y, r, w" epitopes, respectively. The sandwich ELISA was established through using #3-11 (Anti-HBsAg "a" epitope) as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions. CONCLUSION: The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.

[Preparation and characterization of the monoclonal antibodies against human CA15-3 and establishment of a sandwich CLIA system].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 15-3 (CA15-3) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) system for detecting CA15-3 in the human serum. METHODS: BALB/c mice were immunized with human CA15-3 antigen. Spleen cells of the immunized mice were fused with Sp2/0 cells and the positive hybridoma cells were selected and subcloned. The supernatant was taken for purify mAbs using protein A chromatography. The purity, titer, epitope and subtype of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity, repetitiveness and specificity. RESULTS: Five hybridoma cell lines named #3-1-3, #5-2-2, #11-2-2, #12-1-3 and #16-1-3 were obtained respectively, which are of strong positive signal and high secretion. The titers of the mAbs secreted by these hydridomas were above 10(-8); g/mL. All of the mAbs expressed κ light chains, and their heavy chains were as follows: #3-1-3 mAb had IgG2a, #5-2-2 mAb and #12-1-3 mAb had IgG2b, #11-2-2 mAb and #16-1-3 mAb had IgG3. The double-antibody sandwich CLIA system had a good linear relationship between 0.59 U/mL and 300 U/mL. The recovery rate for accuracy was 97.45% and the limit of detection was 0.59 U/mL. The assay was highly linear (correlation coefficient 0.9978) and highly repeatable [coefficient of variation (CV) <10%]. This CLIA system was also highly specific without cross-reactivity to the tumor marker AFP, CEA, CA50, CA19-9 and CA72-4. CONCLUSION: The mAbs against human CA15-3 have been prepared and a sandwich CLIA system for detecting CA15-3 in the human serum has been established successfully, which provides a basis for CA15-3 quantification and clinical application.

[Preparation of monoclonal antibodies against human Apo B100 and establishment of a sandwich ELISA system].

AIM: To prepare the monoclonal antibodies (mAbs) against human Apo B100 and establish a double-antibody sandwich ELISA system for human Apo B100 detection. METHODS: The BALB/c mice were immunized with human Apo B100 antigen. After cell fusion and screening, the hybridoma cells were cultured in serum-free medium and the supernatant was purified to obtain mAbs using protein A. The affinity, subtype, specificity and epitope of the mAbs were characterized and the sandwich ELISA system was established. RESULTS: Four hybridoma cell lines 4-1-2, 4-2-2, 4-3-2 and 4-6-3 were obtained. The affinity of the anti-Apo B100 mAbs was up to 1×109 L/mol and nearly had no cross reaction with other relevant proteins. Linear detection of the sandwich ELISA system using 4-3-2 and 4-6-3 covered a range from (1.3-80) ng/mL, and its sensitivity was 1.24 ng/mL. The intra-assay coefficient of variation (CV) and inter-assay CV were less than 10% and 15%, respectively, and the recovery rate was above 90%. CONCLUSION: The mAbs against human Apo B100 have been prepared and a sandwich ELISA system for human Apo B100 detection has been established successfully, which provide a basis for human Apo B100 detection and disease diagnosis.

[Application of two-color infrared fluorescence for detection of protein phosphorylation].

OBJECTIVE: To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection. METHOD: The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection. RESULTS: LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously. CONCLUSION: Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.

[Progress in study on signal transduction of gametophytic self-incompatibility].

In nature, most self-incompatible flowering plants (angiosperms) show gametophytic self-incompatibility. Although gametophytic self-incompatibility functions can ultimately prevent self-fertilization, flowering plants have adopted different signal transduction pathways to reject self pollen. At present, there are mainly two pathways of signal transduction on gametophytic self-incompatibility. One is the S-RNase-based signal transduction in Solanaceae, Scrophulariaceae, and Rosaceae. The other is the cytosolic free Ca2+ acting as a second messenger in pollen of Papaveraceae. This review highlights the recent progress made towards understanding the signal transduction on gametophytic self-incompatibility.