A Tea Plant (<i>Camellia sinensis</i>) <i>FLOWERING LOCUS C-like</i> Gene, <i>CsFLC1</i>, Is Correlated to Bud Dormancy and Triggers Early Flowering in <i>Arabidopsis</i>

Flowering and bud dormancy are crucial stages in the life cycle of perennial angiosperms in temperate climates. MADS-box family genes are involved in many plant growth and development processes. Here, we identified three MADS-box genes in tea plant belonging to the FLOWERING LOCUS C (CsFLC) family. We monitored CsFLC1 transcription throughout the year and found that CsFLC1 was expressed at a higher level during the winter bud dormancy and flowering phases. To clarify the function of CsFLC1, we developed transgenic Arabidopsis thaliana plants heterologously expressing 35S::CsFLC1. These lines bolted and bloomed earlier than the WT (Col-0), and the seed germination rate was inversely proportional to the increased CsFLC1 expression level. The RNA-seq of 35S::CsFLC1 transgenic Arabidopsis showed that many genes responding to ageing, flower development and leaf senescence were affected, and phytohormone-related pathways were especially enriched. According to the results of hormone content detection and RNA transcript level analysis, CsFLC1 controls flowering time possibly by regulating SOC1, AGL42, SEP3 and AP3 and hormone signaling, accumulation and metabolism. This is the first time a study has identified FLC-like genes and characterized CsFLC1 in tea plant. Our results suggest that CsFLC1 might play dual roles in flowering and winter bud dormancy and provide new insight into the molecular mechanisms of FLC in tea plants as well as other plant species

CsCIPK11-Regulated Metalloprotease CsFtsH5 Mediates the Cold Response of Tea Plants

Photosystem II repair in chloroplasts is a critical process involved in maintaining a plant’s photosynthetic activity under cold stress. FtsH (filamentation temperature-sensitive H) is an essential metalloprotease that is required for chloroplast photosystem II repair. However, the role of FtsH in tea plants and its regulatory mechanism under cold stress remains elusive. In this study, we cloned a FtsH homolog gene in tea plants, named CsFtsH5, and found that CsFtsH5 was located in the chloroplast and cytomembrane. RT-qPCR showed that the expression of CsFtsH5 was increased with leaf maturity and was significantly induced by light and cold stress. Transient knockdown CsFtsH5 expression in tea leaves using antisense oligonucleotides resulted in hypersensitivity to cold stress, along with higher relative electrolyte leakage and lower Fv/Fm values. To investigate the molecular mechanism underlying CsFtsH5 involvement in the cold stress, we focused on the calcineurin B-like-interacting protein kinase 11 (CsCIPK11), which had a tissue expression pattern similar to that of CsFtsH5 and was also upregulated by light and cold stress. Yeast two-hybrid and dual luciferase (Luc) complementation assays revealed that CsFtsH5 interacted with CsCIPK11. Furthermore, the Dual-Luc assay showed that CsCIPK11-CsFtsH5 interaction might enhance CsFtsH5 stability. Altogether, our study demonstrates that CsFtsH5 is associated with CsCIPK11 and plays a positive role in maintaining the photosynthetic activity of tea plants in response to low temperatures

MiR-150 expression promotes myeloid differentiation of CD34+ PBMCs from healthy donors.

<p>Primary CD34+ PBMCs were transduced with pre-miR-150 or empty control lentiviral (ECV) supernatants, sorted for GFP expression, and then assayed by flow cytometry for CD11b, CD34, and CD14 expression or colony formation by colony-forming unit (CFU) assays. (A) Cells were assessed by flow cytometry at the indicated days after cell transduction cultured in the presence of rhSCF, rhIL3, rhIL6, rhGCSF, rhGMCSF (50 ng/ml each), and EPO (2 U/ml). CD11b and CD14 expression increased in both patient samples in the miR-150 vs. control transduced cells, while CD34 did not significantly change. The means from combined patients each run in duplicate experiments are shown; the error bars represent standard deviations (*<i>P≤0</i>.<i>05</i>, for comparisons, Student’s t-test). (B) At day 14 after transduction, miR-150 or ECV transduced CD34+ PBSC were stained with Wright-Giemsa after cytospin preparations and showed morphological evidence of myeloid differentiation, including immature cells (large red arrows), intermediate differentiated cells (large black arrows), and mature myeloid cells (small black arrows). (C) Cells were sorted and plated in triplicate 3 days post transduction in Methocult™ containing 20% lot-tested FBS, 10% BSA, and cytokine concentrations as above. Colonies were counted 12-16 days after plating. The mean colony numbers from triplicate plates are shown for each patient sample (#1 or #2); error bars indicate standard deviations. MiR-150 transduced cells had significantly lower colony numbers compared to control transduced cells, with decreased erythroid colonies (BFU-E and CFU-E), decreased CFU-G and CFU-GM, but increased monocyte/macrophage colonies (CFU-M) (<i>P≤0.05</i> for comparisons, Student’s t-test). (D) Expression of genes associated with myeloid differentiation was assessed in both CD34+ PBSC patient samples transduced with either miR-150 or ECV 9 days after transduction by QPCR. Relative fold-difference in expression for miR-150 vs. ECV cells is displayed for each patient sample; error bars represent standard deviations of technical duplicates.</p

MiR-150 expression promotes myeloid differentiation of primary BC CML patient cells.

<p>Primary BC CML patient samples were transduced with pre-miR-150 or empty control lentiviral (ECV) supernatants, sorted for GFP expression, and then assayed by flow cytometry for CD34 and CD11b expression or colony formation by colony-forming unit (CFU) assays. CD34 expression decreases with myeloid differentiation, whereas CD11b expression increases. (A) CD34+ cells from two BC CML patients were assessed for CD34 and CD11b expression 7 days after transduction in the presence of rhSCF, rhIL3, rhIL6, rhGCSF, and rhGMCSF (50 ng/ml each). Data are shown separately for each patient. Duplicates measurements are reported for patient 1 and triplicate measurements for patient 2. (B) BC CML progenitor cells from patient 1 were sorted and plated in triplicate 5 and 7 days after transduction in Methocult™ containing 20% lot-tested FBS, 10% BSA, and cytokines stated above. Colonies were counted 12-16 days after plating. CFU assays demonstrated decreased myeloid CFUs (CFU-M, CFU-G, and CFU-GM combined) in miR-150 versus control primary BC CML patient cells. Individual colonies were plucked, prepared by cytospin, and stained with Wright-Giemsa to validate morphology. A representative example of cells from a CFU-GM colony (from miR-150 expressing BC CML patient 1 cells) shows monocytes, bands, and granulocytes (top photo) and bands and metamyelocytes (bottom photo). BC CML progenitor cells from patient 2 were sorted and plated in triplicate 4 and 6 days after transduction on Methocult™ as above with the addition of EPO (2 U/ml). A similar decrease in myeloid CFUs was observed. Additionally, BFU-E and CFU-E were also decreased in numbers. The mean colony numbers from triplicate plates are shown; error bars indicate standard deviations (*<i>P≤0</i>.<i>005</i> for comparisons, Student’s t-test).</p

The miR-150 target MYB partially mediates miR-150 induced myeloid differentiation.

<p>(A) HL60 cells were transduced with pre-miR-150, pre-miR-15a/miR-16, empty control lentiviral (ECV) supernatants and then treated with vehicle control (0.1% DMSO), ATRA (1 µM) or TPA (1 ng/mL) for 96 hours and assayed for CD11b expression by flow cytometry. MiR-150 increased CD11b expression in all conditions, including in the absence of differentiating agent or with TPA in contrast to miR-15a/miR-16 and ECV cells. (B) MYB protein expression was assayed in NB4, HL60 and THP-1 miR-150 expressing versus control cell lysates by Western blot with the indicated antibodies. Decreased MYB protein expression was most evident in THP-1 cells expressing miR-150 in the absence of differentiating agent. MYB protein was decreased in both miR-150 and miR-15a/miR-16 expressing HL60 cells after 96 hours of ATRA treatment, but was decreased to the greatest extent in miR-150 expressing cells. (C, D) HL60 and THP-1 cells were transduced with miR-150 (GFP-selectable) and MYB∆3’UTR (YFP-selectable, abbreviated MYB) and control vectors and sorted for double positive YFP and GFP cells. MYB∆3’UTR lacks three miR-150 binding sites. (C) HL60 cells were treated with vehicle control (0.1% DMSO) or the indicated concentrations of ATRA for 96 hours and assayed for CD11b expression by flow cytometry. MYB∆3’UTR expression decreased miR-150 induction of CD11b both in the presence or absence of ATRA. (D) THP-1 cells were treated with vehicle control (0.1% DMSO) or TPA (12.5 ng/mL) for 24 hours, or DMSO or ATRA (1 µM) for 48 hours and then assayed for CD11b expression by flow cytometry. MYB∆3’UTR overexpression significantly decreased CD11b expression in miR-150 expressing cells in either the presence or absence of TPA but not ATRA. For A, C, D two independent experiments, each with three technical replicates, were performed for each condition; the means are shown and the error bars represent standard deviations (*<i>P≤0</i>.<i>05</i>, Student’s t-test).</p

MiR-150 expression promotes myeloid differentiation in AML cell lines.

<p>AML cell lines NB4, PL21, HL60, and THP-1 were transduced with miR-150 or empty control lentiviral (ECV) supernatants, flow sorted for GFP, and assayed 7-21 days post transduction. (A) NB4 cells, an acute promyelocytic leukemia (APL) cell line with the t(15;17) PML-RARA translocation, were treated with the indicated concentrations of ATRA or vehicle control (0.1% DMSO) for 72 hours and assayed by flow cytometry for CD11b expression, displayed as percentage of CD11b positive cells. (B) PL21 cells, an APL cell line with FLT3 ITD, were assayed after 96 hours of ATRA treatment. (C) THP-1 cells, a MLL-AF9 rearranged monocytic AML cell line, were treated with vehicle control (0.1% DMSO) or TPA (12.5 ng/mL) for 24 hours, or DMSO or ATRA (1 µM) for 48 hours and then assayed. (D) HL60 cells, an AML cell line, were assayed after 96 hours of ATRA treatment. For all cell lines assayed, miR-150 expression induced CD11b expression in the absence or presence of TPA or ATRA. For all experiments the means of three independent experiments each performed in triplicate are shown; error bars represent standard deviations (*<i>P≤0</i>.<i>05</i>, ** <i>P≤0.005</i> for comparisons, Student’s t-test). (E) HL60 cells expressing miR-150 displayed increased morphological evidence of differentiation by Wright-Giemsa staining, most notable after exposure to 1 µM ATRA for 96 hours. These features included decreased nuclear to cytoplasmic ratio, lobulated nuclei, and granules. Immature cells are indicated by the large red arrows, intermediate differentiated cells are indicated by the large black arrows and mature myeloid cells by the small black arrows. (F) Quantitation of cells in different stages of myeloid differentiation was determined by Wright-Giemsa stain as described in Methods. Percentage of cells in each stage of differentiation displayed as an average of 10 fields (400 cells counted), error bars indicate standard deviations.</p

MiR-150 expression induces myeloid differentiation in primary AML cells.

<p>Six primary AML patient samples were transduced with pre-miR-150 or empty control lentiviral (ECV) supernatants, sorted for GFP expression, and then assayed by flow cytometry (performed in duplicate) for CD34 and CD11b expression or colony formation by colony-forming unit (CFU) assays. Cells were assessed in presence of rhSCF, rhIL3, rhIL6, rhGCSF, and rhGMCSF (50 ng/ml each) (A) The fold-change increase in CD11b expression in miR-150 expressing cells vs. control cells is displayed individually for 6 AML patient samples. The dotted line indicates the expectation if no change is observed (one sample t-test, P=0.02). (B) Two representative AML patient sample examples of CD11b expression (% positive cells) over time. (C) MiR-150 vs. ECV AML4 patient cells were stained with Wright-Giemsa 12 days after transduction. Arrows indicate immature cells (large red arrows), intermediate differentiated cells (large black arrows), and mature myeloid cells (small black arrows). (D) The following 3-group strategy was used to quantitate cells in various stages of myeloid differentiation: blasts (immature); myelocytes, metamyelocytes, and promonocytes (intermediate); band cells, neutrophils, and monocytes (mature). Percentage of cells in each stage of differentiation displayed as an average of 10 fields (400 cells counted), error bars indicate standard deviations. (E) Expression of genes associated with myeloid differentiation was assessed in AML4 primary cells 9 days after transduction by QPCR. Relative fold-difference in expression for miR-150 vs. ECV cells is displayed; error bars represent standard deviations of technical triplicates.</p

Myeloid differentiation in miR-150 expressing cells is independent of retinoic acid signaling.

<p>(A) HL60 or (B) NB4 miR-150 expressing or control (ECV) cells were co-treated with 100 nM selective RAR antagonist (Ro 41-5253) and 1 ng/ml TPA or 0.1 µM ATRA for 48 hours and assessed for CD11b expression by flow cytometry. Ro decreased ATRA induced CD11b expression in both control and miR-150 transduced cells, but did not block induction of CD11b by TPA treatment or by miR-150 expression in the absence of differentiating agent. For HL60 and NB4 cells two independent experiments, each with three technical replicates, were performed for each condition; the means are shown and the error bars represent standard deviations (*<i>P≤0</i>.<i>05</i>, Student’s t-test).</p

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid’s protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea

Mature miR-150 expression is low in myeloid leukemia cells.

<p>Mature miR-150 expression is decreased in CML and AML cell lines and in primary patient CD34+ progenitor cells from healthy individuals, BC CML and AML samples relative to average miR-150 expression in normal bone marrow (NBM). (A) MiR-150 expression is low or absent in BC CML (K562, LAMA84, and Kcl-22) and AML (HL60, NB4, KG1, PL21, MV4-11, and THP-1) cell lines. Expression is shown as a fold-change relative to miR-150 expression in NBM on a log₁₀ scale as determined by QPCR (performed in duplicate). (B) By QPCR, miR-150 expression was decreased in CD34+ sorted (n=6) vs. unsorted (n=5) NBM. In primary BC CML (n=10) and adult AML (n=22) patient samples, miR-150 expression was significantly decreased compared to unsorted NBM, but not sorted CD34+ NBM. Individual patient samples are shown as fold-change relative to miR-150 expression in NBM on a log₁₀ scale, with means indicated by bars (*<i>P≤0</i>.<i>05, **P≤0.001</i> for comparisons, Student’s t-test). (C) MiR-150 expression was obtained by RNA sequencing for 182 pediatric AML cases and was stratified by risk group based on cytogenetic and molecular abnormalities. Expression is on a log₂ scale, with averages indicated by bars (Student’s t-test used for comparisons).</p