The Systematics, Reproductive Biology, Biochemistry, and Breeding of Sea Buckthorn-A Review.

Both the fruit flesh and seeds of sea buckthorn have multiple uses for medicinal and culinary purposes, including the valuable market for supplementary health foods. Bioactive compounds, such as essential amino acids, vitamins B, C, and E, carotenoids, polyphenols, ursolic acid, unsaturated fatty acids, and other active substances, are now being analyzed in detail for their medicinal properties. Domestication with commercial orchards and processing plants is undertaken in many countries, but there is a large need for improved plant material with high yield, tolerance to environmental stress, diseases, and pests, suitability for efficient harvesting methods, and high contents of compounds that have medicinal and/or culinary values. Applied breeding is based mainly on directed crosses between different subspecies of Hippophae rhamnoides. DNA markers have been applied to analyses of systematics and population genetics as well as for the discrimination of cultivars, but very few DNA markers have as yet been developed for use in selection and breeding. Several key genes in important metabolic pathways have, however, been identified, and four genomes have recently been sequenced.

HrTCP20 dramatically enhance drought tolerance of sea buckthorn (Hippophae rhamnoides L). by mediating the JA signaling pathway.

Sea buckthorn, an important ecological and economical tree species, have remarkable drought and salt resistance. The plant-specific transcription factor TCPs play important roles in plant growth, development, and stress responses. However, in sea buckthorn, the molecular mechanism of TCP proteins and their involvement in drought stress are unknown. Here, we found that the expression of HrTCP20 was significantly up-regulated in sea buckthorn under drought stress. Overexpression of HrTCP20 in Arabidopsis thaliana showed that the superoxide dismutase (SOD), polyphenol oxidase (POD), and chlorophyll (SPAD) content was significantly increased by 1.37 and 1.35 times. However, the malondialdehyde (MDA) content decreased by 0.51 times. Our studies further confirmed that silencing HrTCP20 by virus-induced gene silencing (VIGS) led to a decrease in the content of defense enzymes, relative water content (RWC), and an increase of relative electrical conductivity (REC). Silencing HrTCP20 also caused the jasmonic acid (JA) content to decrease in the VIGS-treated tree. Interestingly, we found that JA accumulation content and the expression of HrLOX2, an essential enzyme for JA synthesis, was significantly inhibited in HrTCP20-silenced sea buckthorn under drought stress. Yeast two-hybrid analysis also showed that HrTCP20 is directly bound to HrLOX2. Taken together, the HrTCP20 transcription factor was a positive regulator in drought resistance of sea buckthorn. Further, our findings will provide comprehensive insights into the forest tree defence system of drought stress.

Explicating genetic diversity based on ITS characterization and determination of antioxidant potential in sea buckthorn (Hippophae spp.).

BACKGROUND: Sea buckthorn (Hippophae) is in the focus of interest mainly for its positive effects on health of both human and animal organisms. Due to the similarities in vegetative morphology, Hippophae species are often misidentified. Therefore, current study was focused on ITS based sequence characterization of sea buckthorn species and comparative biochemical evaluation for its antioxidant properties. METHODS AND RESULTS: DNA was extracted from leaf samples. Primer pairs K-Lab-SeaBukRhm-ITS1F1- K-Lab-SeaBukRhm-ITS1R1 and K-LabSeaBukTib- ITSF1- K-LabSeaBukTib-ITSR1 were used for PCR amplification. The purified PCR products were outsourced for sequencing. Phylogenetic tree was constructed based on neighbor-joining (NJ) method. Moreover, comparison of antioxidant potential of leaves of two sea buckthorn species (Hippophae rhamnoides and Hippophae tibetana) collected from different regions of Ladakh viz., Stakna, Nubra, DRDO Leh and Zanskar was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3- ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and Total antioxidant capacity (TAC) by phosphomolybdenum assays. The present investigation led to the differentiation of two sea buckthorn species viz., H. rhamnoides and H. tibetana based on Internal Transcribed Spacer (ITS) region. Moreover, significant variation was observed in antioxidant potential of leaf extracts collected from different regions. CONCLUSIONS: Primary ITS sequence analysis was found to be powerful tool for identification and genetic diversity studies in sea buckthorn. Leaves of sea buckthorn have pronounced antioxidant properties and can be used in food, neutraceuticals and pharmaceutical industries etc. The current study will pave the way to discover small bioactive molecules responsible for antioxidant and anticancer properties in sea buckthorn.

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers.

Sea buckthorn (Hippophae rhamnoides) is an ecologically and economically important species. Here, we assessed the diversity of 78 accessions cultivated in northern China using 8 agronomic characteristics, oil traits (including oil content and fatty acid composition) in seeds and fruit pulp, and SSR markers at 23 loci. The 78 accessions included 52 from ssp. mongolica, 6 from ssp. sinensis, and 20 hybrids. To assess the phenotypic diversity of these accessions, 8 agronomic fruit traits were recorded and analyzed using principal component analysis (PCA). The first two PCs accounted for approximately 78% of the variation among accessions. The oil contents were higher in pulp (3.46-38.56%) than in seeds (3.88-8.82%), especially in ssp. mongolica accessions. The polyunsaturated fatty acid (PUFA) ratio was slightly lower in the seed oil of hybrids (76.06%) than that of in ssp. mongolica (77.66%) and higher than that of in ssp. sinensis (72.22%). The monounsaturated fatty acid (MUFA) ratio in the pulp oil of ssp. sinensis (57.00%) was highest, and that in ssp. mongolica (51.00%) was equal to the ratio in the hybrids (51.20%). Using canonical correspondence analysis (CCA), we examined the correlation between agronomic traits and oil characteristics in pulp and seeds. Oil traits in pulp from different origins were correlated with morphological groupings (r = 0.8725, p = 0.0000). To assess the genotypic diversity, 23 SSR markers (including 17 loci previously reported) were used among the 78 accessions with 59 polymorphic amplified fragments obtained and an average PIC value of 0.2845. All accessions were classified into two groups based on the UPGMA method. The accessions of ssp. sinensis and ssp. mongolica were genetically distant. The hybrid accessions were close to ssp. mongolica accessions. The 8 agronomic traits, oil characteristics in seed and pulp oils, and 23 SSR markers successfully distinguished the 78 accessions. These results will be valuable for cultivar identification and genetic diversity analysis in cultivated sea buckthorn.

Characterization of the Hippophae rhamnoides WRKY gene family and functional analysis of the role of the HrWRKY21 gene in resistance to abiotic stresses.

Sea buckthorn (Hippophae rhamnoides L.) is a plant with economic and ecological value. It is uniquely capable of growing well under salt and drought stress. WRKY transcription factors play important roles in the ability of plants to resist stress. In this study, 48 HrWRKY genes were identified based on RNA sequencing of H. rhamnoides. Evaluation of expression pattern of HrWRKY1, HrWRKY17, HrWRKY18, HrWRKY21, HrWRKY33-2, HrWRKY40-2, HrWRKY41, and HrWRKY71 suggested that they were involved in abiotic stress. Interestingly, HrWRKY21, one of eight HrWRKY genes, was a positive regulator of abiotic stress tolerance in H. rhamnoides. In addition, most morphological attributes of roots in transgenic Nicotiana tabacum lines (overexpressing HrWRKY21) were also markedly increased compared with the wild-type (WT), including total lengths, specific root lengths, and surface areas. Stress tolerance of transgenic lines was also correlated with higher antioxidant activity (SOD and POD), lower percentage of relative conductivity (REC), and lower activity of malondialdehyde (MDA) under stress conditions. These findings represent a foundation of knowledge about the molecular mechanisms driving resistance to adverse conditions in plants; they are a promising step towards development of tree cultivars with improved tolerance to abiotic stress.

RNA-seq data reveals a coordinated regulation mechanism of multigenes involved in the high accumulation of palmitoleic acid and oil in sea buckthorn berry pulp.

BACKGROUND: Sea buckthorn is a woody oil crop in which palmitoleic acid (C16:1n7, an omega-7 fatty acid (FA)) contributes approximately 40% of the total FA content in berry pulp (non-seed tissue). However, the molecular mechanisms contributing to the high accumulation of C16:1n7 in developing sea buckthorn berry pulp (SBP) remain poorly understood. RESULTS: We identified 1737 unigenes associated with lipid metabolism through RNA-sequencing analysis of the four developmental stages of berry pulp in two sea buckthorn lines, 'Za56' and 'TF2-36'; 139 differentially expressed genes were detected between the different berry pulp developmental stages in the two lines. Analyses of the FA composition showed that the C16:1n7 contents were significantly higher in line 'Za56' than in line 'TF2-36' in the mid-late developmental stages of SBP. Additionally, qRT-PCR analyses of 15 genes involved in FA and triacylglycerol (TAG) biosynthesis in both lines revealed that delta9-ACP-desaturase (ACP-Δ9D) competed with 3-ketoacyl-ACP-synthase II (KASII) for the substrate C16:0-ACP and that ACP-Δ9D and delta9-CoA-desaturase (CoA-Δ9D) gene expression positively correlated with C16:1n7 content; KASII and fatty acid elongation 1 (FAE1) gene expression positively correlated with C18:0 content in developing SBP. Specifically, the abundance of ACP-Δ9D and CoA-Δ9D transcripts in line 'Za56', which had a higher C16:1n7 content than line 'TF2-36', suggests that these two genes play an important role in C16:1n7 biosynthesis. Furthermore, the high expressions of the glycerol-3-phosphate dehydrogenase (GPD1) gene and the WRINKLED1 (WRI1) transcription factor contributed to increased biosynthesis of TAG precursor and FAs, respectively, in the early developmental stages of SBP, and the high expression of the diacylglycerol O-acyltransferase 1 (DGAT1) gene increased TAG assembly in the later developmental stages of SBP. Overall, we concluded that increased ACP-Δ9D and CoA-Δ9D levels coupled with decreased KASII and FAE1 activity is a critical event for high C16:1n7 accumulation and that the coordinated high expression of WRI1, GPD1, and DGAT1 genes resulted in high oil accumulation in SBP. CONCLUSION: Our results provide a scientific basis for understanding the mechanism of high C16:1n7 accumulation in berry pulp (non-seed tissue) and are valuable to the genetic breeding programme for achieving a high quality and yield of SBP oil.

Characterization and identification of ISSR markers associated with oil content in sea buckthorn berries.

Bioactive oils extracted from sea buckthorn (Hippophae rhamnoides) berries contain highly nutritional and medicinal compounds; however, the oil contents of sea buckthorn berries are very low. Thirteen inter-simple sequence repeat (ISSR) primers were used to identify markers associated with oil content of dry pulp in 51 cultivars and lines, which clustered into three major groups based on 137 polymorphic markers. Dry pulp oil contents in 45 cultivars and lines in Group I ranged from 6.6 to 33.1%; these accessions belonged to H. rhamnoides ssp mongolica and its hybrids with H. rhamnoides ssp sinensis. Three lines (H. rhamnoides ssp mongolica) in Group II had high dry pulp oil contents (33.7 to 37.5%), whereas three lines of hybrids in Group III had low dry pulp oil contents (10.9 to 17.5%). The dry pulp oil content of H. rhamnoides ssp mongolica (27.2 ± 0.9%) was higher than that of hybrids (12.0 ± 1.2%) (P < 0.01). Four ISSR markers (881340, 8251000, 817380, and 8071100) had positive association with high dry pulp oil content (P < 0.01) using stepwise multiple regression analysis. The use of these ISSR markers is a potential strategy to select genotypes with high dry pulp oil content and suitable parental combinations for improvement of sea buckthorn berries.

Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites.

Medicinal plants are being widely investigated owing to their ability to produce molecules of therapeutic significance. Isolation of good quality RNA is a tedious but primary step towards undertaking molecular biology experiments. However, medicinal plants are rich in secondary metabolites and not amenable to standard RNA isolation protocols involving Guanidine isothiocyanate (GITC). So an RNA isolation protocol from difficult samples (richer in secondary metabolites) is of highest desiderata. Here we propose a new protocol suitable for isolating RNA from plant tissues rich in secondary metabolites. To standard CTAB (Cetyl Trimethyl Ammonium Bromide) buffer, addition of 2% PVPP (polyvinyl polypyrrolidone) and 350 mM beta-mercaptoethanol was found useful. Use of glacial acetic acid (1M) along with ethanol for precipitation after phenolization and chloroform extraction enhanced the RNA yield. This is the first report of using glacial acetic acid in a CTAB based protocol for the precipitation of RNA. This protocol has been validated in medicinal plant Hippophae rhamnoides vern. seabuckthorn, where standard RNA isolation methods involving GITC and TRIZol extraction buffers failed. The RNA isolated by this method was of good quality as gauged by spectrophotometric readings and denaturing agarose gel electrophoresis. To the best of our knowledge, this RNA isolation protocol has never been published before. The RNA thus obtained could be suitably used for the downstream molecular procedures like Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real Time-PCR, cDNA library construction, etc.