Advances and prospects of orchid research and industrialization.

Orchidaceae is one of the largest, most diverse families in angiosperms with significant ecological and economical values. Orchids have long fascinated scientists by their complex life histories, exquisite floral morphology and pollination syndromes that exhibit exclusive specializations, more than any other plants on Earth. These intrinsic factors together with human influences also make it a keystone group in biodiversity conservation. The advent of sequencing technologies and transgenic techniques represents a quantum leap in orchid research, enabling molecular approaches to be employed to resolve the historically interesting puzzles in orchid basic and applied biology. To date, 16 different orchid genomes covering four subfamilies (Apostasioideae, Vanilloideae, Epidendroideae, and Orchidoideae) have been released. These genome projects have given rise to massive data that greatly empowers the studies pertaining to key innovations and evolutionary mechanisms for the breadth of orchid species. The extensive exploration of transcriptomics, comparative genomics, and recent advances in gene engineering have linked important traits of orchids with a multiplicity of gene families and their regulating networks, providing great potential for genetic enhancement and improvement. In this review, we summarize the progress and achievement in fundamental research and industrialized application of orchids with a particular focus on molecular tools, and make future prospects of orchid molecular breeding and post-genomic research, providing a comprehensive assemblage of state of the art knowledge in orchid research and industrialization.

Genome-Wide Identification and Expression Pattern Analysis of KNOX Gene Family in Orchidaceae.

The establishment of lateral organs and subsequent plant architecture involves factors intrinsic to the stem apical meristem (SAM) from which they are derived. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors that especially act in determining stem cell fate in SAM. Although KNOXs have been studied in many land plants for decades, there is a dearth of knowledge on KNOX's role in Orchidaceae, the largest and most diverse lineage of flowering plants. In this study, a total of 32 putative KNOX genes were identified in the genomes of five orchid species and further designated into two classes (Class I and Class II) based on phylogenetic relationships. Sequence analysis showed that most orchid KNOX proteins retain four conserved domains (KNOX1, KNOX2, ELK, and Homeobox_KN). Comparative analysis of gene structure showed that the exon-intron structure is conserved in the same clade but most orchids exhibited longer intron, which may be a unique feature of Orchidaceae. Cis-elements identified in the promoter region of orchid KNOXs were found mostly enriched in a function of light responsiveness, followed by MeJA and ABA responsiveness, indicative of their roles in modulating light and phytohormones. Collinear analysis unraveled a one-to-one correspondence among KNOXs in orchids, and all KNOX genes experienced strong purifying selection, indicating the conservation of this gene family has been reinforced across the Orchidaceae lineage. Expression profiles based on transcriptomic data and real-time reverse transcription-quantitative PCR (RT-qPCR) revealed a stem-specific expression of KNOX Class I genes and a broader expression pattern of Class II genes. Taken together, our results provided a comprehensive analysis to uncover the underlying function of KNOX genes in Orchidaceae.

[Growth stability of four drought resistant plant species in different regions]. / 四种抗旱植物在不同区域的生长稳定性.

There are abundant germplasm resources of drought resistant trees in China. It is difficult for foresters to evaluate and screen excellent germplasm that is suitable for various drought adverse circumstances. In this study, four tree species from different provenances, namely Amygdalus davi-diana, Prunus sibirica, Salix gordejevii, and Caryopteris mongolica, were used as test materials. Four soil regions, namely Dalad Banner, Siziwang Banner, Kouhezi town and Liujiazi town of Kulun Banner in the Central and Eastern Inner Mongolia Autonomous Region were selected as multi-point experiment sites to analyze the growth and physiological status of different tree species and provenances. The additive main effects and multiplicative interaction model was used to evaluate the regional adaptability and stability of the germplasm. The growth and physiological indices of four tree species differed between provenances and locations. Soil conditions (potassium content, nitrogen content, and pH) and climate conditions (annual average temperature, precipitation, and potential evaportranspiration) in different locations all influenced the growth of different provenance species. Concerning tree species, S. gordejevii and C. mongolica are more adapted to the sandy loam and chestnut soil of Dalad Banner and Siziwang Banner. A. davidiana and P. sibirica L. are more adapted to the loess and aeolian sandy soil of Kouhezi town and Liujiazi town. Concerning tree provenances, A. davidiana of Tuzuo, P. sibirica of Ningcheng and Yuanzhou, S. gordejevii of Lanqi, and C. mongolica of Jingbian displayed higher regional stability and better growth adaptability, indicating their suitability for afforestation in similar areas.

Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution.

As one of the largest families of angiosperms, the Orchidaceae family is diverse. Dendrobium represents the second largest genus of the Orchidaceae. However, an assembled high-quality genome of species in this genus is lacking. Here, we report a chromosome-scale reference genome of Dendrobium chrysotoxum, an important ornamental and medicinal orchid species. The assembled genome size of D. chrysotoxum was 1.37 Gb, with a contig N50 value of 1.54 Mb. Of the sequences, 95.75% were anchored to 19 pseudochromosomes. There were 30,044 genes predicted in the D. chrysotoxum genome. Two whole-genome polyploidization events occurred in D. chrysotoxum. In terms of the second event, whole-genome duplication (WGD) was also found to have occurred in other Orchidaceae members, which diverged mainly via gene loss immediately after the WGD event occurred; the first duplication was found to have occurred in most monocots (tau event). We identified sugar transporter (SWEET) gene family expansion, which might be related to the abundant medicinal compounds and fleshy stems of D. chrysotoxum. MADS-box genes were identified in D. chrysotoxum, as well as members of TPS and Hsp90 gene families, which are associated with resistance, which may contribute to the adaptive evolution of orchids. We also investigated the interplay among carotenoid, ABA, and ethylene biosynthesis in D. chrysotoxum to elucidate the regulatory mechanisms of the short flowering period of orchids with yellow flowers. The reference D. chrysotoxum genome will provide important insights for further research on medicinal active ingredients and breeding and enhances the understanding of orchid evolution.

Physiological characterization, transcriptomic profiling, and microsatellite marker mining of Lycium ruthenicum.

Lycium ruthenicum is a perennial shrub species that has attracted considerable interest in recent years owing to its nutritional value and ability to thrive in a harsh environment. However, only extremely limited transcriptomic and genomic data related to this species can be found in public databases, thereby limiting breeding research and molecular function analysis. In this study, we characterized the physiological and biochemical responses to saline-alkaline mixed stress by measuring photochemical efficiency, chlorophyll content, and protective enzyme activity. We performed global transcriptomic profiling analysis using the Illumina platform. After optimizing the assembly, a total of 68 063 unique transcript sequences with an average length of 877 bp were obtained. Among these sequences, 4096 unigenes were upregulated and 4381 unigenes were down-regulated after saline-alkaline mixed treatment. The most abundant transcripts and over-represented items were assigned to gene ontology (GO) terms or Kyoto Encyclopedia of Genes and the Genomes (KEGG) categories for overall unigenes, and differentially expressed unigenes were analyzed in detail. Based on this set of RNA-sequencing data, a total of 9216 perfect potential simple sequence repeats (SSRs) were identified within 7940 unigenes with a frequency of 1/6.48 kb. A total of 77 primer pairs were synthesized and examined in wet-laboratory experiments, of which 68 loci (88.3%) were successfully amplified with specific products. Eleven pairs of polymorphic primers were verified in 225 individuals from nine populations. The inbreeding coefficient and the polymorphism information content value ranged from 0.011 to 0.179 and from 0.1112 to 0.6750, respectively. The observed and expected heterozygosities ranged from 0.064 to 0.840 and from 0.115 to 0.726, respectively. Nine populations were clustered into three groups based on a genetic diversity study using these novel markers. Our data will be useful for functional genomic investigations of L. ruthenicum and could be used as a basis for further research on the genetic diversity, genetic differentiation, and gene flow of L. ruthenicum and other closely related species.

Overexpression of the poplar NF-YB7 transcription factor confers drought tolerance and improves water-use efficiency in Arabidopsis.

Water deficit is a serious environmental factor limiting the growth and productivity of plants worldwide. Improvement of drought tolerance and efficient water use are significant strategies to overcome this dilemma. In this study, a drought-responsive transcription factor, nuclear factor Y subunit B 7 (PdNF-YB7), induced by osmotic stress (PEG6000) and abscisic acid, was isolated from fast-growing poplar clone NE-19 [Populus nigra × (Populus deltoides × Populus nigra)]. Ectopic overexpression of PdNF-YB7 (oxPdB7) in Arabidopsis enhanced drought tolerance and whole-plant and instantaneous leaf water-use efficiency (WUE, the ratio of biomass produced to water consumed). Overexpressing lines had an increase in germination rate and root length and decrease in water loss and displayed higher photosynthetic rate, instantaneous leaf WUE, and leaf water potential to exhibit enhanced drought tolerance under water scarcity. Additionally, overexpression of PdNF-YB7 in Arabidopsis improved whole-plant WUE by increasing carbon assimilation and reducing transpiration with water abundance. These drought-tolerant, higher WUE transgenic Arabidopsis had earlier seedling establishment and higher biomass than controls under normal and drought conditions. In contrast, Arabidopsis mutant nf-yb3 was more sensitive to drought stress with lower WUE. However, complementation analysis indicated that complementary lines (nf-yb3/PdB7) had almost the same drought response and WUE as wild-type Col-0. Taken together, these results suggest that PdNF-YB7 positively confers drought tolerance and improves WUE in Arabidopsis; thus it could potentially be used in breeding drought-tolerant plants with increased production even under water deficiency.

PdERECTA, a leucine-rich repeat receptor-like kinase of poplar, confers enhanced water use efficiency in Arabidopsis.

Water deficiency causes a dramatic reduction in crop production globally. Breeding crop varieties that are more efficient in their water use is one strategy to overcome this predicament. In this study, a member of the LRR-RLKs family, the Populus nigra × (Populus deltoides × Populus nigra) ERECTA (PdERECTA) gene was cloned. To study the biological functions of PdERECTA, transgenic Arabidopsis plants (35S:PdERECTA) that constitutively expressed the PdERECTA gene were constructed. Overexpression of PdERECTA resulted in early seedling establishment, longer primary roots, and larger leaf areas. Notably, transgenic Arabidopsis overexpressing PdERECTA resulted in enhanced long-term water use efficiency (WUEl), as estimated by the analysis of carbon isotopic discrimination. The WUEl results were supported by the physiological and anatomical results, which included improved photosynthetic rate, decreased transpiration rate, and stomatal density. The transgenic lines have significantly more dry-biomass as compared to the wild type. Since the overexpression of PdERECTA can strongly enhance the water use efficiency in transgenic Arabidopsis plants, PdERECTA could potentially be used in transgenic breeding to improve the water use efficiency.

The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana.

The plant-specific GRAS/SCL transcription factors play diverse roles in plant development and stress responses. In this study, a poplar SCL gene, PeSCL7, was functionally characterized in Arabidopsis thaliana, especially with regard to its role in abiotic stress resistance. Expression analysis in poplar revealed that PeSCL7 was induced by drought and high salt stresses, but was repressed by gibberellic acid (GA) treatment in leaves. Transient expression of GFP-PeSCL7 in onion epidermal cells revealed that the PeSCL7 protein was localized in the nucleus. Transgenic Arabidopsis plants overexpressing PeSCL7 showed enhanced tolerance to drought and salt treatments. The activity of two stress-responsive enzymes was increased in transgenic seedlings. Taken together, these results suggest that PeSCL7 encodes a member of the stress-responsive GRAS/SCL transcription factors that is potentially useful for engineering drought- and salt-tolerant trees.

Molecular characterization of putative vacuolar NHX-type Na(+)/H(+) exchanger genes from the salt-resistant tree Populus euphratica.

The vacuolar NHX-type Na(+)/H(+) exchangers play a key role in salt tolerance in plants. However, little is known about the Na(+)/H(+) exchangers in the salt-resistant tree, Populus euphratica. In this study, we identified six putative vacuolar Na(+)/H(+) exchanger genes from P. euphratica, designated as PeNHX1-6. Real-time polymerase chain reaction indicated that the PeNHX1/3/6 transcripts were abundant compared with the other three PeNHX genes in the three tissues (roots, stems and leaves) examined. After NaCl treatment for 6 h, the transcript levels of PeNHX1-6 were upregulated in the roots. To address the function of PeNHX1-6, complementation studies were performed with the salt-sensitive yeast mutant strain R100, which lacks activity of the endosomal Na(+)/H(+) antiporter NHX1. The results showed that PeNHX1-6 compensates, at least in part, for the function of yeast NHX1. Moreover, PeNHX3 was targeted to the tonoplast when transiently expressed in onion. Together, these results suggest that PeNHX1-6 function as vacuolar Na(+)/H(+) exchangers and that PeNHX products play an important role in the salt resistance of P. euphratica.

Stress responsive zinc-finger protein gene of Populus euphratica in tobacco enhances salt tolerance.

The Populus euphratica stress responsive zinc-finger protein gene PSTZ, which encodes a protein including typical Cys(2)/His(2) zinc finger structure, was isolated by reverse transcription-polymerase chain reaction from P. euphratica. Northern hybridization revealed that its expression was induced under drought and salt stress conditions. To examine its function, cDNA of the PSTZ gene, driven by the cauliflower mosaic virus 35S promoter, was cloned into a plant expression vector pBin438 and introduced into tobacco plants. Transgenic tobacco showed an enhanced salt tolerance, suggesting that PSTZ may play a role in plant responsiveness to salt stress.

[DNA methylation regulating factors in plants].

DNA methylation is an important epigenetic modification and multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. Different methylation sites require different cytosine methyltransferases, which contribute to the modification of chromatin structure and mediate epigenetics with chromatin remodeling enzymes and histone modifying factors. DNA glycosylases can remove DNA methylation and alleviate silencing. The functions and interactions of DNA methylation regulating factors, the establishment, maintenance and removement mechanisms of DNA methylations are reviewed in this paper.

[Cloning and structure analysis of zinc finger protein gene in Populus euphratica Oliv].

Zinc finger proteins belong to a family of nuclear transcription factors which function is to regulate gene expression in both prokaryotic and eukaryotic cells. A pair of primers was designed after analyzing the conservation of salt-tolerant zinc protein Alfin-1 in such diverse plants as alfalfa and Arabidopsis. The zinc finger protein gene is isolated from total RNA with RT-PCR in aquaculture leaves of Populus euphratica . Its full cDNA length is 924bp. Analysis of its amino acid sequence showed it has a typical Cys(2)/His(2) zinc finger structure and a G-rich promoter binding site GTGGGG, starting from position 556. Since transcrptional factors which have the same function show conservation in structure and amino acid sequence of DNA binding region, the structure analysis in this paper indicates the cloned zinc finger protein gene may have functional correlation to Alfin-1.