Transcriptional Regulation of the Acer truncatum B. Response to Drought and the Contribution of AtruNAC36 to Drought Tolerance.

Drought stress is one of the major environmental factors severely restricting plant development and productivity. Acer truncatum B, which is an economically important tree species, is highly tolerant to drought conditions, but the underlying molecular regulatory mechanisms remain relatively unknown. In this study, A. truncatum seedlings underwent a drought treatment (water withheld for 0, 3, 7, and 12 days), after which they were re-watered for 5 days. Physiological indices were measured and a transcriptome sequencing analysis was performed to reveal drought response-related regulatory mechanisms. In comparison to the control, the drought treatment caused a significant increase in antioxidant enzyme activities, with levels rising up to seven times, and relative electrical conductivity from 14.5% to 78.4%, but the relative water content decreased from 88.3% to 23.4%; these indices recovered somewhat after the 5-day re-watering period. The RNA sequencing analysis identified 9126 differentially expressed genes (DEGs), which were primarily involved with abscisic acid responses, and mitogen-activated protein kinase signaling. These DEGs included 483 (5.29%) transcription factor genes from 53 families, including ERF, MYB, and NAC. A co-expression network analysis was conducted and three important modules were analyzed to identify hub genes, one of which (AtruNAC36) was examined to clarify its function. The AtruNAC36 protein was localized to the nucleus and had a C-terminal transactivation domain. Moreover, it bounded specifically to the NACRS element. The overexpression of AtruNAC36 in Arabidopsis thaliana resulted in increased drought tolerance by enhancing antioxidant enzyme activities. These findings provide important insights into the transcriptional regulation mediating the A. truncatum response to drought. Furthermore, AtruNAC36 may be relevant for breeding forest trees resistant to drought stress.

PeGSTU58, a Glutathione S-Transferase from Populus euphratica, Enhances Salt and Drought Stress Tolerance in Transgenic Arabidopsis.

Glutathione S-transferases (GSTs) play a crucial role in responding to abiotic stress and are an important target for research on plant stress tolerance mechanisms. Populus euphratica is a promising candidate species for investigating the abiotic tolerance mechanisms in woody plants. In our previous study, PeGSTU58 was identified as being associated with seed salinity tolerance. In the present study, PeGSTU58 was cloned from P. euphratica and functionally characterized. PeGSTU58 encodes a Tau class GST and is located in both the cytoplasm and nucleus. Transgenic Arabidopsis overexpressing PeGSTU58 displayed enhanced tolerance to salt and drought stress. Under salt and drought stress, the transgenic plants exhibited significantly higher activities of antioxidant enzymes, including SOD, POD, CAT, and GST, compared to the wild-type (WT) plants. Additionally, the expression levels of several stress-responsive genes, including DREB2A, COR47, RD22, CYP8D11, and SOD1 were upregulated in PeGSTU58 overexpression lines compared to those in WT Arabidopsis under salt and drought stress conditions. Furthermore, yeast one-hybrid assays and luciferase analysis showed that PebHLH35 can directly bind to the promoter region of PeGSTU58 and activate its expression. These results indicated that PeGSTU58 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by PebHLH35.

The chloroplast genome sequence and characteristic analysis of Vitex negundo var. heterophylla (Franch.) Rehder.

Vitex negundo var. heterophylla (Franch.) Rehder is a common small shrub in northern China. In order to study the fine nectar characteristics and water and soil conservation characteristics of V. negundo, the analysis of chloroplast genome would provide theoretical basis for economic development and germplasm utilization of V. negundo. The chloroplast genome sequence (accession number MW366787) of V. negundo was accepted by high-throughput sequencing technology using a plant from Jiulongshan, Mentougou District, Beijing, China. The total length of the chloroplast genome is 154,438 bp, and the A, T, C and G content of the whole genome is 30.48, 31.26, 19.42, and 18.84%, respectively. The phylogenetic analysis of 16 Verbenaceae plants (including V. negundo) with Arabidopsis thaliana as the outgroup was carried out by the maximum likelihood method; and the result shows that V. negundo is relatively closed to Vitex rotundifolia.

Characterization of the complete chloroplast genome of basswood (Tilia mongolica maxim.).

Herein, we report the complete chloroplast genome of Tilia mongolica Maxim. from Tiliaceae. The chloroplast genome of T. mongolica is 162,804 bp, with a large single copy region of 91,255 bp, small single copy region of 20,355 bp, and two inverted-repeat regions of 25,597 bp. The chloroplast genome contains 130 genes, including 85 protein-coding, 8 rRNA, and 37 tRNA. The total GC content is 36.46%. The phylogenetic analysis of T. mongolica showed a relatively close relationship with Tilia taishanensis.

Study on a new type of environment-friendly polymer and its preliminary application as soil consolidation agent during tree transplanting.

Transplanting trees with rhizospheric soil is an important way to facilitate tree survival in the process of landscaping and reforestation. Traditional way to prevent looseness of rhizospheric soil is forming soil balls around the roots with bags, boxes or rope wrapping, which is cumbersome, laborious and easy to break. This study is aimed to develop a new type of degradable environment-friendly polymer as soil consolidation agent to facilitate tree transplanting. In this paper, the KGM/CA/PVA ternary blending soil consolidation agent was prepared by using Konjac glucomannan (KGM), chitosan (CA) and polyvinyl alcohol (PVA) as raw materials. Through the verification and evaluation, the clay and sandy soil can be consolidated and formed into soil balls by the ternary blend adhesive, which was convenient for transportation. The preliminary application of the ternary blend adhesive in the transplanting process of sierra salvia, Japanese Spindle (Euonymus japonicus) and Juniperus sabina 'Tamaricifolia' confirmed that the application of soil consolidation agent can effectively solve the problem that the root ball of seedling is easily broken in the process of transplant. And the application of soil consolidation agent has no adverse effect on the growth of transplanted seedlings. The research and development of ternary blending soil consolidation agent and its preliminary application in seedling transplanting will provide a new solution to solve the problem of soil ball breakage in the process of seedling transplanting. This is an important stage in the development of new seedling transplanting technology. Therefore, the research and development of soil consolidation agent is of great significance.

Comparative Transcriptome Analysis Reveals Stem Secondary Growth of Grafted Rosa rugosa 'Rosea' Scion and R. multiflora 'Innermis' Rootstock.

Grafted plant is a chimeric organism formed by the connection of scion and rootstock through stems, so stem growth and development become one of the important factors to affect grafted plant state. However, information regarding the molecular responses of stems secondary growth after grafting is limited. A grafted Rosa plant, with R. rugosa 'Rosea' as the scion (Rr_scion) grafted onto R. multiflora 'Innermis' as the stock (Rm_stock), has been shown to significantly improve stem thickness. To elucidate the molecular mechanisms of stem secondary growth in grafted plant, a genome-wide transcription analysis was performed using an RNA sequence (RNA-seq) method between the scion and rootstock. Comparing ungrafted R. rugosa 'Rosea' (Rr) and R. multiflora 'Innermis' (Rm) plants, there were much more differentially expressed genes (DEGs) identified in Rr_scion (6887) than Rm_stock (229). Functional annotations revealed that DEGs in Rr_scion are involved in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: the phenylpropanoid biosynthesis metabolism and plant hormone signal transduction, whereas DEGs in Rm_stock were associated with starch and sucrose metabolism pathway. Moreover, different kinds of signal transduction-related DEGs, e.g., receptor-like serine/threonine protein kinases (RLKs), transcription factor (TF), and transporters, were identified and could affect the stem secondary growth of both the scion and rootstock. This work provided new information regarding the underlying molecular mechanism between scion and rootstock after grafting.

Role of Aliphatic Chain Characteristics on the Anti-Cracking Properties of Polymer-Modified Asphalt at Low Temperatures.

The anti-cracking properties of polymer-modified asphalt depend largely on the molecular structure of the polymer modifier. However, the mysterious structure-performance relationship is still elusive. In this paper, three kinds of polymers with different chain structures were selected to address this issue. The indices of styrene, trans-butadiene, aliphatic branched-chain, and aliphatic long-chain from the infrared spectrum were used to quantify the functional group compositions of polymer modifiers. Viscoelastic parameters, including relaxation time, dissipation energy ratios, and stiffness were assessed to illustrate the anti-cracking properties of polymer-modified asphalt. Results showed that relaxation time and dissipation energy ratios were mainly determined by the polymer network strength, molecular size, aliphatic chain feature, and the orientations speed of aliphatic chains. The short relaxation time and high dissipation ratio lead to the low stiffness and favorable low-temperature performance of asphalt. The improvement of these performances requires a polymer with high indices of an aliphatic long-chain, styrene, aliphatic branched-chain, and trans-butadiene, respectively. An aliphatic-long chain, aliphatic branched-chain, and trans-butadiene were soft segments in asphalt while styrene was the rigid segment. The soft segments affect the intramolecular friction, orientation, and thermal motion at low temperatures, whereas the rigid segment enhances the strength of polymer networks. Thus, the anti-cracking property of polymer-modified asphalt can be improved by adjusting the ratio of soft and rigid segments in the polymer modifier.

A Stress-Associated Protein, PtSAP13, From Populus trichocarpa Provides Tolerance to Salt Stress.

The growth and production of poplars are usually affected by unfavorable environmental conditions such as soil salinization. Thus, enhancing salt tolerance of poplars will promote their better adaptation to environmental stresses and improve their biomass production. Stress-associated proteins (SAPs) are a novel class of A20/AN1 zinc finger proteins that have been shown to confer plants' tolerance to multiple abiotic stresses. However, the precise functions of SAP genes in poplars are still largely unknown. Here, the expression profiles of Populus trichocarpa SAPs in response to salt stress revealed that PtSAP13 with two AN1 domains was up-regulated dramatically during salt treatment. The ß-glucuronidase (GUS) staining showed that PtSAP13 was accumulated dominantly in leaf and root, and the GUS signal was increased under salt condition. The Arabidopsis transgenic plants overexpressing PtSAP13 exhibited higher seed germination and better growth than wild-type (WT) plants under salt stress, demonstrating that overexpression of PtSAP13 increased salt tolerance. Higher activities of antioxidant enzymes were found in PtSAP13-overexpressing plants than in WT plants under salt stress. Transcriptome analysis revealed that some stress-related genes, including Glutathione peroxidase 8, NADP-malic enzyme 2, Response to ABA and Salt 1, WRKYs, Glutathione S-Transferase, and MYBs, were induced by salt in transgenic plants. Moreover, the pathways of flavonoid biosynthesis and metabolic processes, regulation of response to stress, response to ethylene, dioxygenase activity, glucosyltransferase activity, monooxygenase activity, and oxidoreductase activity were specially enriched in transgenic plants under salt condition. Taken together, our results demonstrate that PtSAP13 enhances salt tolerance through up-regulating the expression of stress-related genes and mediating multiple biological pathways.

Ectopic expression of tea MYB genes alter spatial flavonoid accumulation in alfalfa (Medicago sativa).

Flavonoids are one of the largest secondary metabolite groups, which are widely present in plants. Flavonoids include anthocyanins, proanthocyanidins, flavonols and isoflavones. In particular, proanthocyanidins possess beneficial effects for ruminant animals in preventing lethal pasture bloat. As a major legume forage, alfalfa (Medicago sativa) contains little proanthocyanidins in foliage to combat bloat. In an attempt to improve proanthocyanidin content in alfalfa foliage, we over-expressed two MYB transcription factors (CsMYB5-1 and CsMYB5-2) from tea plant that is rich in proanthocyanidins. We showed that, via targeted metabolite and transcript analyses, the transgenic alfalfa plants accumulated higher levels of flavonoids in stems/leaves than the control, in particular anthocyanins and proanthocyanidins. Over-expression of CsMYB5-1 and CsMYB5-2 induced the expression levels of genes involved in flavonoid pathway, especially anthocyanin/proanthocyanidin-specific pathway genes DFR, ANS and ANR in stems/leaves. Both anthocyanin/proanthocyanidin content and the expression levels of several genes were conversely decreased in flowers of the transgenic lines than in control. Our results indicated that CsMYB5-1 and CsMYB5-2 differently regulate anthocyanins/proanthocyanidins in stems/leaves and flowers. Our study provides a guide for increasing anthocyanin/proanthocyanidin accumulation in foliage of legume forage corps by genetic engineering. These results also suggest that it is feasible to cultivate new varieties for forage production to potentially solve pasture bloat, by introducing transcription factors from typical plants with high proanthocyanidin level.

CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis.

Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a-overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a- and CsMYB5e-overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis.

Over-Expression of Arabidopsis EDT1 Gene Confers Drought Tolerance in Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa L.) is an important legume forage crop with great economic value. However, as the growth of alfalfa is seriously affected by an inadequate supply of water, drought is probably the major abiotic environmental factor that most severely affects alfalfa production worldwide. In an effort to enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced Drought Tolerance 1 (AtEDT1) gene into alfalfa via Agrobacterium-mediated transformation. Compared with wild type plants, drought stress treatment resulted in higher survival rates and biomass, but reduced water loss rates in the transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal size, but reduced stomatal density, and these stomatal changes contributed greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants exhibited larger root systems with larger root lengths, root weight, and root diameters than wild type plants. The transgenic alfalfa plants had reduced membrane permeability and malondialdehyde content, but higher soluble sugar and proline content, higher superoxide dismutase activity, higher chlorophyll content, enhanced expression of drought-responsive genes, as compared with wild type plants. Notably, transgenic alfalfa plants grew better in a 2-year field trial and showed enhanced growth performance with increased biomass yield. All of our morphological, physiological, and molecular analyses demonstrated that the ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in alfalfa. Our study provides alfalfa germplasm for use in forage improvement programs, and may help to increase alfalfa production in arid lands.

Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana.

Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway.

Measurement of serum antibodies against NY-ESO-1 by ELISA: A guide for the treatment of specific immunotherapy for patients with advanced colorectal cancer.

NY-ESO-1 has been identified as one of the most immunogenic antigens; thus, is a highly attractive target for cancer immunotherapy. The present study analyzed the expression of serum antibodies (Abs) against NY-ESO-1 in patients with advanced colorectal cancer (CRC), with the aim of guiding the treatment of NY-ESO-1-based specific-immunotherapy for these patients. Furthermore, the present study was the first to evaluate the kinetic expression of anti-NY-ESO-1 Abs and investigate the possible influencing factors. A total of 239 serum samples from 155 pathologically confirmed patients with advanced CRC (stages III and IV) were collected. The presence of spontaneous Abs against NY-ESO-1 was analyzed using an enzyme-linked immunosorbent assay (ELISA). The results demonstrated that 24.5% (38/155) of the investigated patients were positive for NY-ESO-1-specific Abs. No statistically significant correlations were identified between the expression of anti-NY-ESO-1 Abs and clinicopathological parameters, including age and gender, location, grading, local infiltration, lymph node status, metastatic status and K-ras mutation status (P>0.05). In 59 patients, the kinetic expression of anti-NY-ESO-1 Abs was analyzed, of which 14 patients were initially positive and 45 patients were initially negative. Notably, 16/59 (27.1%) patients changed their expression status during the study period, and the initially positive patients were more likely to change compared with the initially negative patients (85.7 vs. 8.8%; P<0.001). Therefore, monitoring serum Abs against NY-ESO-1 by ELISA is an easy and feasible method. The high expression rate of NY-ESO-1-specific Abs in CRC patients indicates that measuring the levels of serum Abs against NY-ESO-1 may guide the treatment of NY-ESO-1-based specific immunotherapy for patients with advanced CRC.

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes.

Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. No report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus. This work identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abc1, Ubc1, Ubc2, Ubc4, Ubc5, eIF1, eIF2, eIF3, eIF4, EF1, EF2), and used the most suitable combination of reference genes to normalize the expression profiles of seven ROS-scavenging enzyme genes (AmSOD, AmAPX, AmGPX, AmCAT, AmGLR, AmPrx, and AmTrx). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of eIF1 and eIF3 was best for accurate normalization across all the treatments, confirmed by normalizing qPCR data with AmHsp90. In contrast, these data show that Tub1, Abc1, and EF1 are not suitable reference gene candidates. After being normalized against eIF1 and eIF3, the seven ROS-scavenging enzyme genes exhibited differentially up- or down-regulated expression patterns. AmSOD and AmGPX were up-regulated by all four treatments, indicating that they may participate in an anti-oxidative mechanism under abiotic stresses in A. mongolicus. AmCAT exhibited a much higher expression level than AmAPX, AmPrx, and AmGPX, suggesting a principle role in detoxifying excessive H2O2. AmSOD, AmGPX and AmAPX showing the most abundant transcripts under heat, AmCAT and AmGLR under drought, and AmPrx under salt, were observed. Expression patterns of the seven ROS-scavenging enzyme genes suggest different antioxidant protection roles of these genes under abiotic stresses. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.