Comparative analysis of differential gene expression reveals novel insights into the heteroblastic foliage functional traits of Pinus massoniana seedlings.

Pinus massoniana needles, rich in medicinal polysaccharides and flavonoids, undergo heteroblastic foliage, transitioning from primary needles (PN) to secondary needles (SN) during growth, resulting in altered functional traits. Despite its significance, the molecular regulatory mechanisms governing these traits remain unclear. This study employs Iso-Seq and RNA-Seq analyses to explore differentially expressed genes (DEGs) associated with functional traits throughout the main growth season of heteroblastic foliage. Co-expression network analysis identified 34 hub genes and 17 key transcription factors (TFs) influencing light-harvesting antenna, photosystem I and II, crucial in photosynthesis regulation. Additionally, 14 genes involved in polysaccharide metabolism pathways, synthesizing sucrose, glucose, UDP sugars, and xylan, along with four genes in flavonoid biosynthesis pathways, regulating p-coumaroyl-CoA, quercetin, galangin, and myricetin production, exhibited differential expression between PN and SN. Further analysis unveils a highly interconnected network among these genes, forming a pivotal cascade of TFs and DEGs. Therefore, heteroblastic changes significantly impact needle functional traits, potentially affecting the pharmacological properties of PN and SN. Thus, these genomic insights into understanding the molecular-level differences of heteroblastic foliage, thereby establishing a foundation for advancements in the pharmaceutical industry related to needle-derived products.

Regulation of adipogenesis by histone methyltransferases.

Epigenetic regulation is a critical component of lineage determination. Adipogenesis is the process through which uncommitted stem cells or adipogenic precursor cells differentiate into adipocytes, the most abundant cell type of the adipose tissue. Studies examining chromatin modification during adipogenesis have provided further understanding of the molecular blueprint that controls the onset of adipogenic differentiation. Unlike histone acetylation, histone methylation has context dependent effects on the activity of a transcribed region of DNA, with individual or combined marks on different histone residues providing distinct signals for gene expression. Over half of the 42 histone methyltransferases identified in mammalian cells have been investigated in their role during adipogenesis, but across the large body of literature available, there is a lack of clarity over potential correlations or emerging patterns among the different players. In this review, we will summarize important findings from studies published in the past 15 years that have investigated the role of histone methyltransferases during adipogenesis, including both protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs). We further reveal that PRMT1/4/5, H3K4 KMTs (MLL1, MLL3, MLL4, SMYD2 and SET7/9) and H3K27 KMTs (EZH2) all play positive roles during adipogenesis, while PRMT6/7 and H3K9 KMTs (G9a, SUV39H1, SUV39H2, and SETDB1) play negative roles during adipogenesis.

Transcriptomic and targeted metabolomic unravelling the molecular mechanism of sugar metabolism regulating heteroblastic changes in Pinus massoniana seedlings.

Pine seedling leaf characteristics show a distinct transition from primary to secondary needles, known as heteroblastic change. However, the underlying regulatory mechanism is poorly understood. The molecular mechanism of sugar metabolism involved in regulating heteroblastic changes in Pinus massoniana seedlings was investigated via transcriptomics and targeted metabolomics. The results identified 12 kinds of sugar metabolites in the foliage. Three types of sugar accumulated at the highest levels: sucrose, glucose and fructose. Compared to seedlings with only primary needles (PN), the contents of these soluble sugars were lower in seedlings with developing secondary needle buds (SNB). RNA-seq analysis highlighted 1086 DEGs between PN and SNB seedlings, revealing significant enrichment in KEGG pathways including starch and sucrose metabolism, plant hormone signal transduction and amino sugar and nucleic acid sugar metabolism. Combined transcriptomic and metabolomic analysis revealed that HK, MDH, and ATPase could potentially enhance sugar availability by stimulating the glycolytic/TCA cycle and oxidative phosphorylation. These processes may lead to a reduced sugar content in the foliage of SNB seedlings. We also identified 72 transcription factors, among which the expression levels of MYB, WRKY, NAC and C2H2 family genes were closely related to those of DEGs in the sugar metabolism pathway. In addition, we identified alternative splicing (AS) events in one NAC gene leading to two isoforms, PmNAC5L and PmNAC5S. PmNAC5L was significantly upregulated, while PmNAC5S was significantly downregulated in SNB seedlings. Overall, our results provide new insights into how sugar metabolism is involved in regulating heteroblastic changes in pine seedlings.

Complete Chloroplast Genome Sequence of Fagus longipetiolata Seemen (Fagaceae): Genome Structure, Adaptive Evolution, and Phylogenetic Relationships.

Fagus longipetiolata Seemen is a deciduous tree of the Fagus genus in Fagaceae, which is endemic to China. In this study, we successfully sequenced the cp genome of F. longipetiolata, compared the cp genomes of the Fagus genus, and reconstructed the phylogeny of Fagaceae. The results showed that the cp genome of F. longipetiolata was 158,350 bp, including a pair of inverted repeat (IRA and IRB) regions with a length of 25,894 bp each, a large single-copy (LSC) region of 87,671 bp, and a small single-copy (SSC) region of 18,891 bp. The genome encoded 131 unique genes, including 81 protein-coding genes, 37 transfer RNA genes (tRNAs), 8 ribosomal RNA genes (rRNAs), and 5 pseudogenes. In addition, 33 codons and 258 simple sequence repeats (SSRs) were identified. The cp genomes of Fagus were relatively conserved, especially the IR regions, which showed the best conservation, and no inversions or rearrangements were found. The five regions with the largest variations were the rps12, rpl32, ccsA, trnW-CCA, and rps3 genes, which spread over in LSC and SSC. The comparison of gene selection pressure indicated that purifying selection was the main selective pattern maintaining important biological functions in Fagus cp genomes. However, the ndhD, rpoA, and ndhF genes of F. longipetiolata were affected by positive selection. Phylogenetic analysis revealed that F. longipetiolata and F. engleriana formed a close relationship, which partially overlapped in their distribution in China. Our analysis of the cp genome of F. longipetiolata would provide important genetic information for further research into the classification, phylogeny and evolution of Fagus.

Downregulation of SUV39H1 and CITED2 Exerts Additive Effect on Promoting Adipogenic Commitment of Human Mesenchymal Stem Cells.

Human adipogenesis is the process through which uncommitted human mesenchymal stem cells (hMSCs) differentiate into adipocytes. Through a siRNA-based high-throughput screen that identifies adipogenic regulators whose expression knockdown leads to enhanced adipogenic differentiation of hMSCs, two new regulators, SUV39H1, a histone methyltransferase that catalyzes H3K9Me3, and CITED2, a CBP/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 were uncovered. Both SUV39H1 and CITED2 are normally downregulated during adipogenic differentiation of hMSCs. Further expression knockdown induced by siSUV39H1 or siCITED2 at the adipogenic initiation stage significantly enhanced adipogenic differentiation of hMSCs as compared with siControl treatment, with siSUV39H1 acting by both accelerating fat accumulation in individual adipocytes and increasing the total number of committed adipocytes, whereas siCITED2 acting predominantly by increasing the total number of committed adipocytes. In addition, both siSUV39H1 and siCITED2 were able to redirect hMSCs to undergo adipogenic differentiation in the presence of osteogenic inducing media, which normally only induces osteogenic differentiation of hMSCs in the absence of siSUV39H1 or siCITED2. Interestingly, simultaneous knockdown of both SUV39H1 and CITED2 resulted in even greater levels of adipogenic differentiation of hMSCs and expression of CEBPα and PPARγ, two master regulators of adipogenesis, as compared with those elicited by single gene knockdown. Furthermore, the effects of co-knockdown were equivalent to the additive effect of individual gene knockdown. Taken together, this study demonstrates that SUV39H1 and CITED2 are both negative regulators of human adipogenesis, and downregulation of both genes exerts an additive effect on promoting adipogenic differentiation of hMSCs through augmented commitment.

Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells.

BACKGROUND: Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation. CONCLUSIONS: RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells

BACKGROUND: Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation. CONCLUSIONS: RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Resveratrol exerts dosage and duration dependent effect on human mesenchymal stem cell development.

Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs.

Herpes simplex virus type-1 (HSV-1) entry into human mesenchymal stem cells is heavily dependent on heparan sulfate.

Hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type-1 (HSV-1). The purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hMSCs) to HSV-1 infection and identify the major entry receptor. Productive virus infection in hMSCs was confirmed by replication and plaque formation assays using a syncytial HSV-1 KOS (804) virus. To examine the significance of entry receptors, RT-PCR and antibody-blocking assays were performed. RT-PCR data showed the expression of gD receptors: nectin-1, 3-O sulfotransferase-3 (3-OST-3), and HVEM. Antibody-blocking assay together with heparinase treatment suggested an important role for HS and 3-O-sulfated heparan sulfate (3-OS HS), but not nectin-1 or HVEM, in mediating HSV-1 entry and spread in hMSCs. Taken together, our results provide strong evidence demonstrating that HSV-1 is capable of infecting hMSCs and HS and 3-OS HS serve as its entry receptors during this process.

A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells.

Tissue-specific (or adult) stem/progenitor cells are regarded as the source for normal tissue homeostasis and tissue repair. They also provide tremendous promise for regenerative medicine because of their capacity to proliferate and differentiate into a variety of mature cell types. Human mesenchymal stem cells (hMSCs) can differentiate into osteocytes, adipocytes, chondrocytes, muscle cells, and neurons. However, the molecular mechanisms underlying these differentiation processes are poorly understood. We screened a synthetic siRNA library targeting 5,000 human genes to identify the endogenous repressors of osteogenic specification, which when silenced could initiate differentiation of hMSCs into osteoblasts. This screen yielded 53 candidate suppressors, and 12 of those were further confirmed for their dynamic roles in suppressing osteogenic specification in hMSCs. Furthermore, cAMP was identified to play opposing roles in osteogenesis vs. adipogenesis. This study provides a basis for further elucidation of the genetic network controlling osteogenesis and, potentially, the molecular rationale for treating bone diseases.

Genomic studies in stem cell systems.

Stem cell research holds promise for future regenerative medicine. In recent years, much effort has been devoted to our understanding of stem cell self-renewal and differentiation at the molecular level. Important studies that have applied various genomic approaches, including genomics, functional genomics and proteomics, to gain molecular insights into stem cell development, are reviewed.

HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis.

We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM) development in Arabidopsis thaliana. Mutants have fused sepals and reduced organ numbers in all four whorls, especially in the 2nd (petal) and 3rd (stamen) whorls. han meristems can become flatter or smaller than in the wild type. HAN encodes a GATA-3-like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the meristem and its newly initiated organ primordia and at the boundaries between different floral whorls. It is also expressed in vascular tissues, developing ovules and stamens, and in the embryo. han interacts strongly with clavata (clv) mutations (clv1, clv2, and clv3), resulting in highly fasciated SAMs, and we find that WUS expression is altered in han mutants from early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is normally required for establishing organ boundaries in shoots and flowers and for controlling the number and position of WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of meristem activity, suggesting that HAN is involved in controlling cell proliferation and differentiation.

Floral homeotic genes are targets of gibberellin signaling in flower development.

Gibberellins (GAs) are a class of plant hormones involved in the regulation of flower development in Arabidopsis. The GA-deficient ga1-3 mutant shows retarded growth of all floral organs, especially abortive stamen development that results in complete male sterility. Until now, it has not been clear how GA regulates the late-stage development of floral organs after the establishment of their identities within floral meristems. Various combinations of null mutations of DELLA proteins can gradually rescue floral defects in ga1-3. In particular, the synergistic effect of rga-t2 and rgl2-1 can substantially restore flower development in ga1-3. We find that the transcript levels of floral homeotic genes APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) are immediately upregulated in young flowers of ga1-3 upon GA treatment. Using a steroid-inducible activation of RGA, we further demonstrated that these floral homeotic genes are transcriptionally repressed by RGA activity in young flowers whereas the expression of LEAFY (LFY) and APETALA1 (AP1) is not substantially affected. In addition, we observed the partial rescue of floral defects in ga1-3 by overexpression of AG. Our results indicate that GA promotes the expression of floral homeotic genes by antagonizing the effects of DELLA proteins, thereby allowing continued flower development.

Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeoboxes.

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5' cluster positions were generally dominant over more 3' homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development.