Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis

MicroRNAs (miRNAs) are small, noncoding RNAs that can play crucial regulatory roles in eukaryotes by targeting mRNAs for silencing. To test whether miRNAs play roles in the regulation of wood development in tree species, we isolated small RNAs from the developing xylem of Populus trichocarpa stems and cloned 22 miRNAs. They are the founding members of 21 miRNA gene families for 48 miRNA sequences, represented by 98 loci in the Populus genome. A majority of these miRNAs were predicted to target developmental- and stress/defense-related genes and possible functions associated with the biosynthesis of cell wall metabolites. Of the 21 P. trichocarpa miRNA families, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental expression patterns, suggesting that even conserved miRNAs may have different regulatory roles in different species. Most unexpectedly, the remaining 10 miRNAs, for which 17 predicted targets were experimentally validated in vivo, are absent from the Arabidopsis genome, suggesting possible roles in tree-specific processes. In fact, the expression of a majority of the cloned miRNAs was upregulated or downregulated in woody stems in a manner consistent with tree-specific corrective growth against tension and compression stresses, two constant mechanical loads in trees. Our results show that plant miRNAs can be induced by mechanical stress and may function in one of the most critical defense systems for structural and mechanical fitness.     

Identification and Characterization of ABA-Responsive MicroRNAs in Rice

MicroRNAs(miRNAs) are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition.The phytohormone abscisic acid(ABA) is essential for plant development and adaptation to abiotic and biotic stresses.In Arabidopsis,miRNAs are implicated in ABA functions.However,ABA-responsive miRNAs have not been systematically studied in rice.Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant,Osabal.Of these,13 were confirmed by stem-loop RT-PCR.Among them,miR1425-5P,miR169 a,miR169n,miR390-5P,miR397 a and miR397 b were up-regulated,but miR162 b reduced in expression in Osabal.The targets of these 13 miRNAs were predicted and validated by gene expression profiling.Interestingly,the expression levels of these miRNAs and their targets were regulated by ABA.Cleavage sites were detected on 7 of the miRNA targets by 5'-Rapid Amplification of cDNA Ends(5'-RACE).Finally,miR162 b and its target OsTREl were shown to affect rice resistance to drought stress,suggesting that miR162 b increases resistance to drought by targeting OsTREl.Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.     

Sex-specific carbon and nitrogen partitioning under N deposition in Populus cathayana

Key message

Females of Populus cathayana allocated increased N to soluble proteins, while males keep N allocation pattern unchanged under N enrichment.

Abstract

In our study, Populus cathayana as a model species is employed to detect the sex-specific responses in growth, photosynthetic nitrogen (N) use efficiency (PNUE), carbon (C) and N partitioning when exposed to 0, 7.5 and 15 g N m^?2 year^?1 on the basis of local N deposition level. Our results showed that females had higher responses in photosynthesis and growth than males when exposed to N deposition, and also exhibited higher PNUE than males when exposed to high level of N deposition, suggesting that plasticity in the females may enhance the capacity of young seedlings to acquire resources. Moreover, we found that females mainly allocated increased N to soluble proteins and detergent-soluble proteins, but not to cell-wall proteins, while males maintained original partitioning pattern of N and also accumulated excessive N in the form of free amino acids. Females also allocated more fraction of leaf N to carboxylation (P _C), bioenergetics (P _B), and then more fraction of leaf N to all components of photosynthetic machinery (P _T) than males under high level of N deposition. Such sex-specific N allocation strategy may correlate with sex-related PNUE. These results indicated that there is a higher critical N demand in females, and females could use N nutrient more efficiently than males under high N deposition. We also found that higher shift from starch to soluble sugars, such as sucrose, occurred in females than in males under N enrichment. By contrast, excessive accumulation of starch and non-structural carbohydrate in females relative to males was observed under control conditions, which might inhibit female photosynthesis rate. Accordingly, we suggested that the different leaf C and N partitioning patterns could explain the sex-specific responses in growth. Therefore, females may obtain advantageous position in the process of intraspecific competition when exposed to high level of N deposition because they have higher light capture (total leaf area) ability and utilization efficiency (PNUE) than the males that confer the ability for fast growth and thus are likely to be more responsive to N enrichment. Our results suggested that it could be important to look at the stronger growth response of the females over the males under N enrichment at both the leaf and the plant scale.     

香杨的组织培养和植株再生

1 植物名称 香杨（Populus koreana Rehd.）。 2 材料类别 顶芽和腋芽。 3 培养条件 以1/2MS为基本培养基。（1）不定芽诱导培养基：1/2MS＋6-BA0.5mg·L^-1（单位下同）＋NAA0.1;（2）不定芽增殖培养基：1/2MS＋6-BA1.0＋NAA0.1;（3）生根培养基：1/2MS＋6-BA0.1＋NAA0.1。     

毛果杨 NLP 基因家族生物信息学分析与鉴定简

NLP基因家族是一类特殊的转录因子,豆科植物根瘤的形成依赖于该基因家族的存在,在非豆科植物中具有调节植物硝酸盐吸收以及同化的功能。通过对毛果杨（Populus trichocarpa）基因组的生物信息学分析,共鉴定出14个毛果杨NLP基因家族成员,这些成员具有低亲水性的特点,基因结构保守,都含有RWP-RK以及PB1两个保守结构域。通过细胞定位预测,所有成员都定位在细胞核中。直系同源与旁系同源进化分析显示,NLP基因家族成员在漫长的进化过程中经历了严格的选择。染色体定位分析表明,毛果杨NLP基因家族成员坐落在毛果杨9条染色体之上,成员数量的扩增来自于杨柳科染色体自身的扩增事件。芯片数据分析结果显示,NLP基因家族成员在嫩叶,根和雄花中表达,部分基因在木质部以及种子萌发过程之中表达,但所有成员均不在成熟叶片中表达。     

Sex-related differences in physiological and ultrastructural responses of Populus cathayana to Ni toxicity

We used Populus cathayana, a native species with an extensive distribution in northern, central, and southwestern China, as a model species to detect the sex-specific differences in photosynthetic capacity, ultrastructure, nitrogen (N) metabolism, and nickel (Ni) accumulation and distribution in response to Ni stress. Exposure to 100 μM Ni²⁺ in a hydroponic system for 1 month significantly decreased the pigment content and the photosynthetic rate, caused visible impairment in cellular organelle structure, and induced obvious disturbance and imbalance in the N content of female plants, while male plants suffered a lower negative influence on all the above measured parameters. However, males accumulated a higher Ni concentration in both leaves and roots than females, while the transportation ratio of Ni from roots to shoots in males was slightly lower than that in females. Our results, therefore, suggest that males have a better tolerance capacity and a greater ability to remediate Ni-polluted soil than females. This greater tolerance capacity in males might be highly correlated with the better maintenance of N balance and more effective physiological detoxification responses (such as the response to proline) under Ni stress. The differences between the sexes in tolerance capacity to heavy metals should be verified after performing a field investigation using adult trees as materials in the future study.     

Sex-Related Responses of Populus cathayana Shoots and Roots to AM Fungi and Drought Stress

We investigated the impact of drought and arbuscular mycorrhizal (AM) fungi on the morphological structure and physiological function of shoots and roots of male and female seedlings of the dioecious plant Populus cathayana Rehder. Pot-grown seedlings were subjected to well watered or water-limiting conditions (drought) and were grown in soil that was either inoculated or not inoculated with the AM fungus Rhizophagus intraradices. No significant differences were found in the infection rates between the two sexes. Drought decreased root and shoot growth, biomass and root morphological characteristics, whereas superoxide radical (O₂–) and hydrogen peroxide content, peroxidase (POD) activity, malondialdehyde (MDA) concentration and proline content were significantly enhanced in both sexes. Male plants that formed an AM fungal symbiosis showed a significant increase in shoot and root morphological growth, increased proline content of leaves and roots, and increased POD activity in roots under both watering regimes; however, MDA concentration in the roots decreased. By contrast, AM fungi either had no effect or a slight negative effect on the shoot and root growth of female plants, with lower root biomass, total biomass and root/shoot ration under drought. In females, MDA concentration increased in leaves and roots under both watering regimes, and the proline content and POD activity of roots increased under drought conditions; however, POD activity significantly decreased under well-watered conditions. These findings suggest that AM fungi enhanced the tolerance of male plants to drought by improving shoot and root growth, biomass and the antioxidant system. Further investigation is needed to unravel the complex effects of AM fungi on the growth and antioxidant system of female plants.     

去叶对青杨雌雄植株生长和非结构性碳水化合物的影响

近年来,森林食叶害虫在全世界呈爆发趋势.树木的非结构性碳水化合物(NSC)如何响应叶片损失对其生长和生存至关重要.雌雄异株植物在维持森林生态系统稳定性方面扮演着重要角色.然而,目前对该类植物性别之间如何响应去叶的研究还比较少.本文以我国重要的经济和生态恢复树种青杨(Populus cathayana)为研究材料,比较了...     

Higher soil salinity causes more physiological stress in female of Populus cathayana cuttings

Dioecious plant species represent an major component of terrestrial ecosystems. Little is known about sex-specific responses to soil salinity. Populus cathayana Rehd, which is a dioecious, deciduous tree, was employed as a test species in our study. In a semi-controlled environment, physiological responses to salinity were investigated in male and female P. cathayana cuttings, which were subjected to two salt regimes: 0 and 80 mM NaCl added to the Hoagland’s solution for one month growth. Relative to the control, the saline treatment significantly decreased net photosynthetic rate (P_n), transpiration (E), stomatal conductance (g_s), carotenoids (Caro), chlorophyll a (Chl a), total chlorophyll (TC) and catalase (CAT) activity, but increased Na⁺, Ca²⁺, K⁺, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and carbon isotope composition (δ¹³C) in both sexes. Different sensitivity to saline conditions between males and females was detected. With higher soil salinity, females exhibited lower P_n, Chl a, TC, Chl a/b, Ca²⁺, Ca²⁺/Na⁺, K⁺/Na⁺, SOD and CAT activities but higher Na⁺ and MDA content than males. However, there were no significant differences in these traits (except for SOD and CAT activities) detected in the control group. Our results indicated that males may be more tolerate to salinity than females, with females having lower gas exchanges, chlorophyll pigments, antioxidant enzyme activities, K⁺/Na⁺ ratio and water use efficiency (WUE) than males.     

青杨雌雄叶片气孔分布及气体交换的异质性差异

利用网格和二维成图的方法对青杨雌雄叶片各区域的气孔分布及气体交换特性进行了测定.结果表明:1)除气孔密度(SD)外,雌雄植株在气孔长度(SL)、宽度(SW)和比值(SR)方面具有显著差异(P=0.000,P=0.000和P=0.002).雌株的SL和SW分别比雄株的高51.86%和67.06%,而SR则比雄株的低11.46%.从雌株和雄株的叶面分布来看,SD均为叶中>叶尖>叶基,SL均为叶尖>叶中>叶基,SW的最小值同在叶基部,但最大值分别在叶中和叶尖部.雌株的SR表现为叶基>叶尖>叶中,雄株却正好相反.2)在净光合速率(Pn)上雌株明显低于雄株.雌株的Pn叶基最低(Pn值介于2.00～3.00?μmol m-2 s-1),叶尖最高(Pn值介于8.00～9.00?μmol m-2 s-1),总体上表现出沿叶基到叶尖逐渐增大的趋势.雄株的Pn在叶面的分布也有差异,但总体规律不明显.3)从叶面各区间的气体交换来看,雌株的蒸腾速率(Tr)和气孔导度(Gs)变化不大,雄株的Tr和Gs呈现叶尖和叶中部高于叶基部的明显趋势.雌株的胞间CO2浓度(Ci)为叶基>叶中>叶尖,气孔限制值(Ls)与此相反.而雄株的Ci在叶面的变化较大,Ls呈现沿叶尖至叶基方向逐步上升的趋势.上述结果表明,青杨雌雄叶片在气孔分布及气体交换特性上不仅存在显著的性别差异,而且具有明显的叶面区域异质性.     

Identification and Characterization of Novel Antibacterial Peptides from Skin Secretions of Euphlyctis cyanophlyctis

In this study, we extracted and purified antimicrobial peptides (AMPs) secreted from skin of Euphlyctis cyanophlyctis using reverse phase-high performance liquid chromatography. Three AMPs were isolated from skin secretions of this frog and sequenced using tandem mass spectrometry. The purified peptides were named buforin-EC (1875.05 ± 0.5 Da), cyanophlyctin (2347.50 ± 0.5 Da) and temporin-ECa (1013.33 ± 0.5 Da). Multiple alignments and homology search showed that buforin-EC, cyanophlyctin and temporin-ECa had a homology of 71.43, 47.1, and 69.23% to buforin II, brevinin-2EC, and temporin-1CSc, respectively. Antimicrobial tests demonstrated that our peptides have a great antimicrobial effect on both gram-positive and gram-negative bacteria. The results indicated that they have an overall minimum inhibitory concentration (MIC) below 13 μM against E. coli. No hemolysis was observed in around of their MIC values. In conclusion, skin secretions of E. cyanophlyctis contain a novel class of AMPs with the proper characteristics.     

Identification and Characterization of Novel Lipophilic Antimicrobial Peptides Derived from Naturally Occurring Proteins

Microbes are increasingly developing defensive mechanisms against known drugs via mutations. There are signs of emergence of superbugs immune to most known antibiotics available. The need for a new class of drugs to counteract this problem is of paramount importance for continued general well being of mankind. A new class of drugs, antimicrobial peptides, has not been fully exploited primarily due to high cytotoxicity, poor lipophilicity preventing systemic distribution and stability. We have synthesised 9-amino acid residue cationic peptides RH01 and RH02 lipidated with myristoyl and octyl groups respectively. These peptides exhibited potent antimicrobial activity and low cytotoxicity. The lipopeptide RH01 has antimicrobial activity against a broad range of microorganisms including bacteria, yeast and filamentous fungi with greatest activity toward Gram-positive bacteria, including S. aureus MRSA stain, MIC’s ranging between 2–8 μM. The MIC for Gram-negative bacteria was higher ranging from between 30–250 μM. RH01 also had antimicrobial activity towards fungi showing good activity against the pathogenic yeast Candida albicans but was less active towards the filamentous fungi Aspergillus niger. The antimicrobial activity of RH01 as a measure of Ki₍₅₀₎ for E. coli and S. aureus was 35–60 μM and 3–7 μM, respectively. In-house data showed the compound is bactericidal even at higher bacteria concentration. The octylated lipopeptide RH02 has similar activities towards S. aureus (3.3 μM) and E coli (53.3 μM) as the myristolated RH01. There was no haemolytic activity of the lipopeptide RH01 towards human blood. Acute intravenous toxicity study in mice showed that both RH01 and RH02 induced no macroscopic abnormalities at their highest non-lethal dose of 75 mg/kg and 150 mg/kg bodyweight, respectively.Australian Peptide Conference Issue.