Genome-wide identification of Aux/IAA and ARF gene families reveal their potential roles in flower opening of Dendrobium officinale.

BACKGROUND: The auxin indole-3-acetic acid (IAA) is a vital phytohormone that influences plant growth and development. Our previous work showed that IAA content decreased during flower development in the medicinally important orchid Dendrobium officinale, while Aux/IAA genes were downregulated. However, little information about auxin-responsive genes and their roles in D. officinale flower development exists. RESULTS: This study validated 14 DoIAA and 26 DoARF early auxin-responsive genes in the D. officinale genome. A phylogenetic analysis classified the DoIAA genes into two subgroups. An analysis of cis-regulatory elements indicated that they were related by phytohormones and abiotic stresses. Gene expression profiles were tissue-specific. Most DoIAA genes (except for DoIAA7) were sensitive to IAA (10 µmol/L) and were downregulated during flower development. Four DoIAA proteins (DoIAA1, DoIAA6, DoIAA10 and DoIAA13) were mainly localized in the nucleus. A yeast two-hybrid assay showed that these four DoIAA proteins interacted with three DoARF proteins (DoARF2, DoARF17, DoARF23). CONCLUSIONS: The structure and molecular functions of early auxin-responsive genes in D. officinale were investigated. The DoIAA-DoARF interaction may play an important role in flower development via the auxin signaling pathway.

Shoot organogenesis and somatic embryogenesis from leaf and petiole explants of endangered Euryodendron excelsum.

Euryodendron excelsum H.T. Chang is a rare and endangered woody plant endemic to China. It is very important to conserve and propagate this species from extinction. In this study, leaves and petioles from the axillary shoots in vitro were used as explants to culture on the different plant growth regulator (PGR) woody plant medium (WPM) and establish an efficient shoot proliferation and plant regeneration system. WPM supplemented with 1.0 mg/L 2,4-D induced callus dedifferentiated into buds and somatic embryos on various media,including PGR-free WPM. However, only adventitious shoots formed on WPM with 1.0 mg/L of cytokinins such as 6-benzyladenine (BA), kinetin (KIN) or thidiazuron (TDZ). When another cytokinin, zeatin, was used, somatic embryos were induced directly from From cut surface of these explants. Adventitious roots could be induced from both explants on WPM with 1.0 mg/L α-naphthaleneacetic acid (NAA). Somatic embryos cultured in PGR-free WPM or WPM with 0.2 mg/L NAA developed roots. Plantlets derived from somatic embryos were transferred to a peat: sand (1:1, v/v) substrate, and showed survival rates of 64.3% at 30 days and 54.6% at 90 days. Callus clumps with adventitious shoot buds that were transferred to WPM containing 1.0 mg/L BA and 0.2 mg/L NAA generated a mean 3.3 multiple shoots. Callus-derived shoots regenerated and rooted successfully (100%) on agar-free vermiculite-based WPM with 0.5 µM NAA after 30 d. Plantlets transplanted to peat soil: vermiculite (1:1, v/v) displayed the highest survival (96.7%) after three months.

Rice histone deacetylase HDA704 positively regulates drought and salt tolerance by controlling stomatal aperture and density.

MAIN CONCLUSION: HDA704 enhances drought and salt tolerance via stomata-regulated mechanism. HDA704 negatively regulates stomatal aperture and density, repressing the transcription of DST and ABIL2 by histone deacetylation modification. Drought and salinity can damage crop growth and reduce yield. Stomata play an important role in abiotic stress tolerance. In this study on rice, we identified the RPD3/HDA1-type histone deacetylase HDA704 as a positive regulatory factor in drought and salt tolerance. HDA704 was induced by drought and salt stresses. Overexpression of HDA704 in transgenic rice promoted stomatal closure, decreased the number of stomata and slowed down the rate of water loss, consequently resulting in increased drought and salt tolerance. By contrast, knockdown of HDA704 in transgenic rice decreased stomatal closure and accelerated the rate of water loss, leading to decrease drought and salt tolerance. We detected the transcript expression of DST (Drought and Salt Tolerance) and ABIL2 (Abscisic Acid-insensitive Like2), which positively regulate stomatal aperture and density in rice. Our results showed that HDA704 directly binds to DST and ABIL2, repressing their expression via histone deacetylation modification. Collectively, these findings reveal that HDA704 positively regulates drought and salt tolerance by repressing the expression of DST and ABIL2. Our findings provide a new insight into the molecular mechanisms of stomata-regulated abiotic stress tolerance of plants.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album.

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Cloning, characterization, and functional analysis of acetyl-CoA C-acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA synthase genes in Santalum album.

Sandalwood (Santalum album L.) is famous for its unique fragrance derived from the essential oil of heartwood, whose major components are santalols. To understand the mechanism underlying the biosynthesis of santalols, in this study, we cloned two related genes involved in the mevalonate pathway in S. album coding for acetyl-CoA C-acetyl transferase (AACT) and 3-hydroxy-3-methyglutary-CoA synthase (HMGS). These genes were characterized and functionally analyzed, and their expression profiles were also assessed. An AACT gene designated as SaAACT (GenBank accession No. MH018694) and a HMGS gene designated as SaHMGS (GenBank accession No. MH018695) were successfully cloned from S. album. The deduced SaAACT and SaHMGS proteins contain 415 and 470 amino acids, and the corresponding size of their open-reading frames is 1538 bp and 1807 bp, respectively. Phylogenetic trees showed that the SaAACT protein had the closest relationship with AACT from Hevea brasiliensis and the SaHMGS proteins had the highest homology with HMGS from Siraitia grosvenorii. Functional complementation of SaAACT and SaHMGS in a mutant yeast strain deficient in these proteins confirmed that SaAACT and SaHMGS cDNA encodes functional SaAACT and SaHMGS that mediate mevalonate biosynthesis in yeast. Tissue-specific expression analysis revealed that both genes were constitutively expressed in all examined tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) of S. album, both genes showing highest expression in roots. After S. album seedlings were treated with 100 µM methyl jasmonate, the expression levels of SaAACT and SaHMGS genes increased, suggesting that these genes were responsive to this elicitor. These studies provide insight that would allow further analysis of the role of genes related to the sandalwood mevalonate pathway in the regulation of biosynthesis of sandalwood terpenoids and a deeper understanding of the molecular mechanism of santalol biosynthesis.

Axillary shoot proliferation and plant regeneration in Euryodendron excelsum H. T. Chang, a critically endangered species endemic to China.

Euryodendron excelsum H. T. Chang is a single-type, rare and endangered woody plant unique to China. In this study, young stems were used as explants and cultured on Woody Plant Medium (WPM) supplemented with 5.0 µM 6-benzyladenine (BA), were subcultured for more than 15 times over a total of more than 3 years and finally an efficient axillary shoot proliferation and plantlet regeneration system was established in which one shoot could proliferate an average of 5.1 axillary shoots every 2 months on the medium supplemented with 5.0 µM BA and 0.5 µM α-naphthaleneacetic acid (NAA). Shoots rooted at a moderate frequencies (50.1%) on agarized WPM supplemented with 0.5 µM NAA but 100% of shoots rooted in agar-free vermiculite-based WPM after culture for 2 months. Plantlets, when transplanted to peat soil: vermiculite (1:1), showed the highest 95.1% survival within 1 month.

Development, progress and future prospects in cryobiotechnology of Lilium spp.

Lilium is one of the most popular flower crops worldwide, and some species are also used as vegetables and medicines. The availability of and easy access to diverse Lilium genetic resources are essential for plant genetic improvements. Cryopreservation is currently considered as an ideal means for the long-term preservation of plant germplasm. Over the last two decades, great efforts have been exerted in studies of Lilium cryopreservation and progress has been made in the successful cryopreservation of pollen, seeds and shoot tips in Lilium. Genes that exist in Lilium, including those that regulate flower shape, color and size, and that are resistant to cold stress and diseases caused by fungi and viruses, provide a rich source of valuable genetic resources for breeding programs to create novel cultivars required by the global floriculture and ornamental markets. Successful cryopreservation of Lilium spp. is a way to preserve these valuable genes. The present study provides updated and comprehensive information about the development of techniques that have advanced Lilium cryopreservation. Further ideas are proposed to better direct future studies on Lilium cryobiotechnology.

Changes in DNA methylation pattern of apple long-term in vitro shoot culture and acclimatized plants.

DNA methylation is a process of epigenetic modification that can alter the functionality of a genome. Using whole-genome bisulfite sequencing, this study quantify the level of DNA methylation in the epigenomes of two diploid apple (Malus x domestica) scion cultivars ('McIntosh' and 'Húsvéti rozmaring') derived from three environmental conditions: in vivo mother plants in an orchard, in vitro culture, and acclimatized in vitro plants. The global DNA methylation levels were not dependent on the source of plant material, and the average level of DNA methylation was 49.77%, 34.65% and 8.77% in CpG, CHG and CHH contexts, respectively. Significant differences in DNA methylation were identified in 586 (specifically 334, 201 and 131 in CpG, CHG and CHH contexts, respectively) out of 45,116 genes, including promoter and coding sequences. These were classified as differentially methylated genes (DMGs). This is a 1.3% difference in the level of DNA methylation of genes in response to a change in the environment. Differential methylation was visualised by MA plots and functional genomic maps were established for biological processes, molecular functions and cellular components. When the DMGs were considered, in vitro tissue culture resulted in the highest level of methylation, but it was lower in acclimatized in vitro plants which was similar to that in the mother tree. Methylation patterns of the two scions differed, indicating cultivar-specific epigenetic regulation of gene expression during adaptation to various environments. After selecting genes that displayed differences larger than ±10% in CpG and CHG contexts, or larger than ±1.35% in the CHH context from among the DMGs, they were annotated in Blast2 GO v5.1.12 for Gene Ontology. DMGs identified as MD07G1113000 (protein transport), MD08G1041600 (extracellular space), MD09G1054800 (phosphatidic acid binding), and MD10G1265800 (not annotated) were methylated in all three contexts in in vitro shoots. These DNA methylation results suggest that epigenetic changes may contribute to the adaptation of apple to environmental changes by modifying the epigenome and thereby gene expression.

Transcriptome and chemical analysis reveal putative genes involved in flower color change in Paeonia 'Coral Sunset'.

The flower color of Paeonia 'Coral Sunset' and 'Pink Hawaiian Coral' changes from coral to pink to pale yellow during flowering, which confers high ornamental value to these two cultivars. However, the molecular mechanism underlying flower color change is still unclear. In this study, flavonoids in petals of Paeonia 'Coral Sunset' and 'Pink Hawaiian Coral' at seven flowering stages were analyzed to explore the effects of the flavonoid component on changes in flower color. In addition, four cDNA libraries of 'Coral Sunset' during the critical blooming stages were constructed and the transcriptome was sequenced to investigate the molecular mechanism underlying changes to flower color. Two anthocyanins (cyanidin-3,5-di-O-glucoside and peonidin-3,5-di-O-glucoside) were detected in both cultivars. Total anthocyanin content in both cultivars accumulated continuously from stages 1-3 and then decreased sharply. Correlation analysis showed that the change in flower color from coral to pink to pale yellow is due to a significant decrease in anthocyanin content. A total of 91,583 Unigenes were obtained in 'Coral Sunset', 33,962 (37.08%) of which were annotated to major databases. Based on the enrichment analysis of differentially expressed genes, eight structural genes (CHS, F3H, F3'H, FLS, DRF, ANS, ANR and UFGT) and 13 transcription factors (five MYB, three bHLH, one WD40, one HY5, one PIF3, one COP1 and two PHY) related to flavonoid biosynthesis were screened. The qRT-PCR results were generally consistent with the high-throughput sequencing results. This research will provide a foundation to clarify the mechanisms underlying changes in flower color of herbaceous peony.

Molecular Characterization of Cyclamen Species Collected from Different Parts of Turkey by RAPD and SRAP Markers.

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Aydin, Mugla, Izmir, Denizli, Kahramanmaras, Osmaniye, Eskisehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.

Methods for genetic transformation in Dendrobium.

KEY MESSAGE: The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Somatic Embryogenesis in Two Orchid Genera (Cymbidium, Dendrobium).

The protocorm-like body (PLB) is the de facto somatic embryo in orchids. Here we describe detailed protocols for two orchid genera (hybrid Cymbidium Twilight Moon 'Day Light' and Dendrobium 'Jayakarta', D. 'Gradita 31', and D. 'Zahra FR 62') for generating PLBs. These protocols will most likely have to be tweaked for different cultivars as the response of orchids in vitro tends to be dependent on genotype. In addition to primary somatic embryogenesis, secondary (or repetitive) somatic embryogenesis is also described for both genera. The use of thin cell layers as a sensitive tissue assay is outlined for hybrid Cymbidium while the protocol outlined is suitable for bioreactor culture of D. 'Zahra FR 62'.

In vitro propagation of Paphiopedilum orchids.

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.

Magnetic fields: how is plant growth and development impacted?

This review provides detailed insight on the effects of magnetic fields on germination, growth, development, and yield of plants focusing on ex vitro growth and development and discussing the possible physiological and biochemical responses. The MFs considered in this review range from the nanoTesla (nT) to geomagnetic levels, up to very strong MFs greater than 15 Tesla (T) and also super-weak MFs (near 0 T). The theoretical bases of the action of MFs on plant growth, which are complex, are not discussed here and thus far, there is limited mathematical background about the action of MFs on plant growth. MFs can positively influence the morphogenesis of several plants which allows them to be used in practical situations. MFs have thus far been shown to modify seed germination and affect seedling growth and development in a wide range of plants, including field, fodder, and industrial crops; cereals and pseudo-cereals; grasses; herbs and medicinal plants; horticultural crops (vegetables, fruits, ornamentals); trees; and model crops. This is important since MFs may constitute a non-residual and non-toxic stimulus. In addition to presenting and summarizing the effects of MFs on plant growth and development, we also provide possible physiological and biochemical explanations for these responses including stress-related responses of plants, explanations based on dia-, para-, and ferromagnetism, oriented movements of substances, and cellular and molecular changes.

How Authorship is Defined by Multiple Publishing Organizations and STM Publishers.

The most important part of a biomedical scientific manuscript is undeniably the research data. Yet, scientists generate and validate that data, culminating, in most cases, in a scientific manuscript. Thus, authorship, specifically the contributions and attributed responsibilities of the authors, remains a central issue in science publishing. This article examines the definitions of authorship as defined by four publishing organizations--the Committee on Publication Ethics (COPE), the Council of Scientific Editors (CSE), the International Committee of Medical Journal Editors (ICMJE), and World Association of Medical Editors (WAME)-and 15 science, technology, and medicine (STM) publishers. The objective is to understand whether there is consistency among definitions. Five of these STM publishers rely specifically on the ICMJE definitions of authorship, while 12/15 are COPE members. The clarity, logic, realism, feasibility, and enforceability of these definitions will be discussed. Our analysis reveals that authorship definitions are inconsistent among the 15 STM publishers. Scientists have the inherent right to determine who is an author of an article according to the ethical guidelines of their institutes, but these may differ from the guidelines indicated by publishers, while editors and publishers have the right to verify authorship.

Dendrobium micropropagation: a review.

Dendrobium is one of the largest and most important (ornamentally and medicinally) orchid genera. Tissue culture is now an established method for the effective propagation of members of this genus. This review provides a detailed overview of the Dendrobium micropropagation literature. Through a chronological analysis, aspects such as explant, basal medium, plant growth regulators, culture conditions and final organogenic outcome are chronicled in detail. This review will allow Dendrobium specialists to use the information that has been documented to establish, more efficiently, protocols for their own germplasm and to improve in vitro culture conditions based on the optimized parameters detailed in this review. Not only will this expand the use for mass propagation, but will also allow for the conservation of important germplasm. Information on the in vitro responses of Dendrobium for developing efficient protocols for breeding techniques based on tissue culture, such as polyploidization, somatic hybridization, isolation of mutants and somaclonal variants and for synthetic seed and bioreactor technology, or for genetic transformation, is discussed in this review. This is the first such review on this genus and represents half a decade of literature dedicated to Dendrobium micropropagation.

Chemical composition, antioxidant activity and in vitro antibacterial activity of Achillea wilhelmsii C. Koch essential oil on methicillin-susceptible and methicillin-resistant Staphylococcus aureus spp.

The present study investigated the chemical composition of the essential oil (EO) from aerial parts (flowering stage) of Achillea wilhelmsii C. Koch by GC-MS. In addition, the antioxidant activity of the EO as well as its antimicrobial activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) strains was tested. Antioxidant activity was measured by the ability of the EO to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals while the antimicrobial activity was assessed by the disc-diffusion method. In total, 52 compounds were recognized, accounting for 97.33 % of the EO. The main compounds in the EO were carvacrol (22.49 %), dihydrocarvone (13.23 %), linalool (12 %), 1,8-cineol (11.42 %), camphene (8.31 %), thymol (5.28 %), camphor (3.71 %), pulegone (2.82 %) α-terpineol (2.11 %), bornyl acetate (1.14 %), and farganol (1.01 %). The EC50 value of the EO was 0.01 and 0.08 mg/mL for the antioxidant and DPPH-scavenging ability, respectively. A. wilhelmsii EO affected methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA, but the impact was more effective on MSSA.

Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor.

BACKGROUND: The formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation. RESULTS: Analysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets. CONCLUSIONS: An extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.