Purification and Identification of Antifreeze Proteins in Ammopiptanthus mongolicus

The conventional protein chromatography technique was adopted to purify the antifreeze proteins (AFPs) from the leaves of Ammopiptanthus mongolicus (Maxim.) Cheng f. Two bands on native PAGE gel showed thermal hysteresis activity, one was band B1, whose thermal hysteresis was 0.46 ℃ at 8 g/L, which showed two bands (67 kD, 21 kD) on SDS_PAGE gel; the other was B3, whose thermal hysteresis was 0.45 ℃ at 10 g/L, and it contained only a single protein (39.8 kD). Both B1 and B3 are not glycoproteins, because neither do they interact with Shiff_reagent, nor show ultraviolet characteristics of a typical glycoprotein.

Effects of Drought Stress on the Photoprotection in Ammopiptanthus mongolicus Leaves

Ammopiptanthus mongolicus (Maxim.) Cheng f. is one of the evergreen shrubs in the desert region of China. In midday its leaves bear photon flux density over 1 500 μmol·m -2 ·s -1 at natural habitat. They show the obvious phenomenon of photoinhibition. For the study of the effects of drought stress on the major protective mechanism against strong light in A. mongolicus leaves, the diurnal variations of photosynthetic rate and chlorophyll fluorescence parameters were investigated under natural conditions with portable photosynthetic measurement system (CIRAS_1) and portable fluorometer (MFMS_2). The experimental results showed that, under normal and drought stress conditions,the net photosynthetic rate ( Pn ), the primary maximum photochemical efficiency of PSⅡ ( Fv/Fm ) and the quantum efficiency of noncyclic electron transport of PSⅡ ( Φ PSⅡ ) decreased obviously at noon (Figs.2,3A,4B). In comparison with plants under normal condition, under drought stress minimal chlorophyll fluorescence ( Fo ) decreased at first and then increased (Fig.3A), non_photochemical quenching ( NPQ ) quickly increased and sustained at a higher level (Fig.4B). This indicated that the major photoprotective mechanism of A. mongolicus leaves was the xanthophyll cycle_dependent thermal energy dissipation under normal condition, while under drought stress, the major photoprotective mechanism was both the xanthophyll cycle_dependent thermal energy dissipation and the reversible inactivation of PSⅡ reaction center.

Effects of the Extract of Ammopiptanthus mongolicus cheng f. (JA1) on Induction of Apoptosis of HepG2 in vitro and Its Molecular Mechanisms

Objective To study the effects and the mechanisms of extract from a leguminous plant （Ammopiptanthus mongolicus cheng f.） （JAl ） in northwest China on inducing apoptosis and inhibiting proliferation of HepG2 hepatocarcinoma cell in vitro. Methods The HepG2 cell line was used as target cells. The effect of 3A 1 on HepG2 cell growth was detected by microculture tetrazolium assay （MTr）, flow cytometry assay, DNA agarose gel electrophoresis and transmission electronic microscopy. The expressive effect of the wt-p53 in HepG2 cells was analyzed with p53 protein test-reagent. Results JAl not only had significant anti-proliferative effects depending upon time and dosage, but also induced apoptosis of HepG2 cells. Apoptotic typical morphological changes were observed in JAl-treated HepG2 cells under transmission electronic microscope, ＂Sub-G 1＂ phase peak occurred in flow cytometry and DNA ＂ladder＂ was found in DNA agarose gel electrophoresis. The expression of the wt-p53 increased in vitro, and 3Al-treated HepG2 and the positive cell percentage of the wt-p53 protein also increased. Conclusions JAl could obviously induce apoptosis and inhibit proliferation of HepG2 cells in vitro, and these effects are closely related with the increase of wt-p53 expression. JAl can be used as a good source of medicinal plant for the treatment of hepatocarcinoma.

Effects of cold-hardening on compatible solutes and antioxidant enzyme activities related to freezing tolerance in Ammopiptanthus mongolicus seedlings

Cold acclimation is associated with many metabolic changes that lead to an increase of freezing tolerance. In order to investigate the biochemical process of cold acclimation in Ammopiptanthus mongolicus, seedlings were acclimated at 2℃ under 16-h photoperiod （150 μmol·m^-2·s^-1 photosynthetically active radiation） for 14 d. Freezing tolerance in seedlings increased after 14 d of cold-hardening. Contents of protein, proline and solute carbohydrate in cotyledon increased after cold acclimation. Patterns of isozymes of superoxide dismutase （SOD）, peroxidase, catalase and polyphenol oxidase （PPO） were investigated. The activities of SOD, peroxidase and PPO in cold acclimated plants were increased during cold-hardening. We deduced that compatible solutes and antioxidant enzymes play important roles in development of freezing tolerance during cold acclimation in this evergreen woody plant.

Cold acclimation induced accumulation of phenolic compounds and freezing tolerance in Ammopiptanthus mongolicus

Ammopiptanthus mongolicus, the only freezing tolerant evergreen broad-leaved shrub, local species of the Alashan desert, northwest sand area of China, can survive -30℃ or even lower temperature in winter. In the present study, the secondary products phenolics in A. mongolicus cotyledons were determined to study the effects of phenolics on cold tolerance. Cytochemical localization of phenolics in cotyledon cells was observed by electron microscopy and the content of phenolic compounds was assayed by spectrophotometric measurement. The results showed that the freezing tolerance ofA. mongolicus seedlings increased after acclimation at 2℃ for 14 days, which accompanied the increase of the content of phenolic compounds in cotyledons. Cytochemical observation showed that phenolic deposits were mainly localized in vacuoles and in close proximity to tonoplast, and also in the cytoplasm. The amount and the size of phenolics droplets increased obviously in cytoplasm and vacuoles after cold acclimation, predominantly aggregated along membranes of vacuoles and tonoplast. No phenolic deposits were found in cell walls. As hydrogen- or elec- tron-donating agents, phenolics may protect plant cells against reactive oxygen species formed during chilling or freezing stress and improve the freezing tolerance of cold-acclimated A. mongolicus seedlings.

Molecular cloning and functional analyses of low-temperature induced genes from Ammopiptanthus mongolicus

Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene isolation.Through differential expression analysis induced by low-temperature,thirteen up-regulated cDNAs were identified.One of them,AmEBP1,(accession number:DQ519359)confers enhanced cold-tolerance to both transgenic E.coli and transgenic Arabidopsis.Results suggest that AmEBP1 can stimulate the synthesis of ribosome and the dephosphyration of the α-subunit of initiation factor 2(eIF2α),and subsequently promote the translation process.By which the transgenic plants obtained increased cold-resistant ability.

A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and droughttolerance mechanisms

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms.Ammopiptanthus mongolicus(Leguminosae)is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions;however,its drought-tolerance mechanisms remain poorly understood.Here,we report the assembly of a reference-grade genome for A.mongolicus,describe its evolutionary history within the legume family,and examine its drought-tolerance mechanisms.The assembled genome is 843.07 Mb in length,with 98.7%of the sequences successfully anchored to the nine chromosomes of A.mongolicus.The genome is predicted to contain 47611 protein-coding genes,and 70.71%of the genome is composed of repetitive sequences;these are dominated by transposable elements,particularly longterminal-repeat retrotransposons.Evolutionary analyses revealed two whole-genome duplication(WGD)events at 130 and 58 million years ago(mya)that are shared by the genus Ammopiptanthus and other legumes,but no species-specific WGDs were found within this genus.Ancestral genome reconstruction revealed that the A.mongolicus genome has undergone fewer rearrangements than other genomes in the legume family,confirming its status as a"relict plant".Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed,both under normal conditions and in response to polyethylene glycol-induced dehydration.Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress,suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A.mongolicus.Ectopic expression of AmERF2,an ethylene response factor unique to A.mongolicus,can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants,demonstrating the potential for application of A.mongolicus genes in crop improvement.

干旱胁迫下枯草芽孢杆菌(Bacillus subtilis)对柠条(Caragana korshinskii)和沙冬青(Ammopiptanthus mongolicus)种子萌发及幼苗生长的影响

土壤微生物具有丰富的功能多样性和极强的环境适应性,微生物固沙成为防沙治沙技术研发的新兴领域。枯草芽孢杆菌(Bacillus subtilis)是常用的微生物固沙材料,但已有研究多聚焦于其防风固沙效果,较少注意其对沙生植物定植和生长生理的影响。为揭示枯草芽孢杆菌对沙生植物种子萌发和幼苗生长生理特征的影响,以柠条(Caragana korshinskii)和沙冬青(Ammopiptanthus mongolicus)为研究对象,开展室内盆栽试验,对比分析不同干旱梯度和菌剂浓度下种子萌发、幼苗生长和生理生化特征。结果表明:(1)重度干旱显著抑制柠条种子萌发和幼苗生长(P<0.05),中度、重度干旱显著抑制沙冬青株高生长(P<0.05);菌剂处理显著促进柠条和沙冬青种子萌发(P<0.05),高浓度菌剂显著促进柠条幼苗生长(P<0.05),但菌剂抑制了沙冬青冠幅生长。(2)枯草芽孢杆菌对柠条和沙冬青叶绿素的产生有促进效果,柠条在不同干旱胁迫条件下的最适菌剂浓度不同;干旱胁迫增加柠条和沙冬青幼苗丙二醛、脯氨酸含量,施用菌剂柠条的丙二醛含量显著低于对照组(P<0.05),除中度干旱胁迫外,沙冬青菌剂处理组丙二醛和脯氨酸含量均显著低于对照组(P<0.05)。(3)干旱胁迫和菌剂处理交互作用对柠条和沙冬青的种子萌发和幼苗生长不存在极显著影响(P>0.01),但对二者叶绿素、丙二醛和脯氨酸含量存在极显著影响(P<0.01)。枯草芽孢杆菌可以通过影响两种沙生植物生理生化活动间接影响种子萌发和幼苗生长,提高植物抗逆性。

Water use strategy of Ammopiptanthus mongolicus community in a drought year on the Mongolian Plateau

Aims In desert ecosystems,water is a restricting factor for plant growth and vegetation dynamics.The relatively stable water source from deep soil profile or groundwater is important for plant survival during drought.Understanding water use strategy of endangered species,in desert ecosystem is essential for their conservation and restoration such as Ammopiptanthus mongolicus on the Mongolian Plateau.Methods The stable isotope method ofδD andδ^(18)O was used to examine the main water sources of A.mongolicus and two companion shrubs,e.g.Artemisia ordosica and Artemisia xerophytica.The contribution of different water sources to each species was calculated by IsoSource model.Leafδ13C was used to compare long-term water use efficiency of three shrubs.Soil moisture and root distribution of three shrubs was measured to explain plant water use strategy.Important Findings The results showed that A.mongolicus relied on groundwater and 150–200 cm deep soil water,with the former contributing to almost half of its total water source.Artemisia ordosica mainly used 150–200 cm deep soil water,but also used shallow soil water within 100 cm in summer and autumn.Artemisia xerophytica mainly used 150–200 cm deep soil water and groundwater,with the latter contributing to about 30%–60%of its total water source.The three shrubs had dimorphic or deep root systems,which are in accord with their water sources.The WUE in the evergreen shrub A.mongolicus was higher than in two deciduous Artemisia shrubs,which may be an adaptive advantage in desert ecosystem.Therefore,groundwater is an important water source for the endangered shrub A.mongolicus in a drought year on Mongolian Plateau.Ammopiptanthus mongolicus and two Artemisia shrubs competed for deep soil water and groundwater.

Genome-wide Identification of microRNAs that Respond to Drought Stress in Seedlings of Tertiary Relict Ammopiptanthus mongolicus

microRNAs(miR NAs) are important post-transcriptional regulators of gene expression in plant abiotic stress responses. Ammopiptanthus mongolicus is a typical evergreen woody xerophyte, which makes it an ideal model system for studying drought tolerance in woody plants. The response of mi RNAs to drought stress is still unknown in this plant. In this research, we obtained 34 695 665 raw reads from two small RNA libraries constructed from control and drought-treated A. mongolicus seedlings by the Illumina deep sequencing technology. Length analysis revealed that reads of 20, 21, and 24 nucleotides accounted for the majority of the small RNAs in the two libraries. Sequence analyses identified 65 conserved mi RNA sequences from 190 members of 31 mi RNA families and 20 potential novel mi RNAs from 17 families that were differentially expressed between the two libraries. The expression patterns of 25 of these mi RNAs were significantly different in the two libraries, but only am-mi R4 a-c was downregulated during drought stress, the other 24 were upregulated. The expression trends determined by the Hi Seq sequencing and by q RT-PCR were similar. Furthermore, 35 target genes for the conserved mi RNA were markedly changed and 52 target genes for the potential novel mi RNAs were predicted and their functions were annotated by computational analysis. Our results provide new molecular evidence for understanding the molecular mechanisms of drought response and tolerance in A. mongolicus.

Overexpression of AmDUF1517 enhanced tolerance to salinity, drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmD UF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmD UF1517 cotton displayed significantly higher antioxidant enzyme（superoxide dismutase（SOD）, peroxidase（POD）, catalase（CAT）, and glutathione S-transferase（GST）） activity and less reactive oxygen species（ROS） accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.

Analysis of sparteines in the seeds of Ammopiptanthus mongolica

Seven alkaloids were isolated from the seeds of Ammopiptanthus mongolica by thin layer chromatography and silica gel column chromatography,and the chemical structures of five alkaloids,17-oxosparteine,b-isosparteine,3a-hydroxysparteine,sparteine,and 3b-hydroxysparteine were identified by 1H nuclear magnetic resonance(NMR)and electron ionization mass spectrum(EIMS).

Leaf-surface wax of desert plants as a potential lubricant additive

Using an MFT-R4000 tester at room temperature,the leaf-surface wax of two desert plants,Ammopiptanthus mongolicus(AM)and Reaumuria soongorica(RS),was extracted and evaluated for its potential as a lubricant additive in polyalphaolefin(PAO)for steel-steel contact.Gas chromatography-mass spectrometry analysis was performed to identify the composition of the AM leaf-surface wax,and scanning electron microscopy and X-ray photoelectron spectroscopy were used to investigate its friction mechanisms.The results suggest that the leaf-surface wax could successfully reduce the friction and wear of steel-steel sliding pairs compared with PAO containing molybdenum dithiocarbamate additives.AM,in particular,showed high-performance wear resistance and friction-reducing properties.Its excellent tribological properties were attributed to the wax composition of leaf-surface fatty acids,alcohol,and esters.