Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na⁺ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na⁺ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na⁺ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.     

Bracelet salt glands of the recretohalophyte Limonium bicolor: Distribution,morphology, and induction

Halophytes complete their life cycles in saline environments. The recretohalophyte Limonium bicolor has evolved a specialized salt secretory structure,the salt gland, which excretes Na⁺to avoid salt damage. Typical L. bicolor salt glands consist of 16 cells with four fluorescent foci and four secretory pores. Here, we describe a special type of salt gland at the base of the L. bicolor leaf petiole named bracelet salt glands due to their beaded-bracelet-like shape of blue auto-fluoresc...     

盐胁迫对二色补血草种子萌发的影响

以二色补血草(Limonium bicolor)种子为试验材料,用不同浓度的NaCl、Na2SO4及二者质量比为1:1的混合盐在其种子萌发过程中进行胁迫,调查盐胁迫对种子萌发的影响,并测定萌发过程中种子蛋白质含量、淀粉酶活力及还原糖和总糖含量的变化.结果表明:盐胁迫使二色补血草种子的发芽势、发芽指数和发芽率明显降低,而且随着盐浓度的增鲻加,抑制作用增强;3种盐对种子萌发的抑制作用强度依次为NaCl>混合盐>Na2SO4;二色补血草种子耐盐适宜范围为Na2SO4 1.2%、混合盐0.8%、NaCl 0.5%,耐盐半致死浓度为Na2SO4 2.1%、混合盐1.4%、NaCl 1.1%,耐盐极限浓度为Na2SO4 3.5%、混合盐2.4%、NaCl 2.0%.盐处理后不萌发的种子转入蒸馏水后重新萌发的试验结果表明,盐分胁迫主要是通过渗透效应,抑制种子中蛋白质和总糖的分解,进而延缓或抑制了种子的萌发.     

An efficient autofluorescence method for screening Limonium bicolor mutants for abnormal salt gland density and salt secretion

With the spread of saline soils worldwide, it has become increasingly important to understand salt-tolerant mechanisms and to develop halophytes with increased salt tolerance. Limonium bicolor is a typical recretohalophyte and has a typical salt excretory structure in the epidermis called the salt gland. A method that can be used to screen a large population of L. bicolor mutants for altered salt gland density and altered salt secretion is needed but is currently unavailable. Leaves of 1-month-old L. bicolor seedlings were processed by three traditional methods [epidermal peel, nail impression, and clearing/differential interference contrast microscope (clearing/DIC) method] and a fluorescence method (fluorescence microscopic examination of cleared leaves). With the fluorescence method, the autofluorescence of salt glands under UV excitation (330–380 nm) was easily distinguished with the least labor and time. The fluorescence method was used to screen ~ 10,000 seedlings (which grew from gamma-irradiated seeds). Four mutants with reduced salt gland density and 15 with increased salt gland density were obtained. Both kinds of mutants will be useful for the isolation of genes involved in salt gland development and salt secretion in L. bicolor and other halophytes. The fluorescence method was also successfully used to observe the salt glands of Aegialitis rotundifolia and the stomata and trichomes of Arabidopsis. The fluorescence method described here will be useful for examining plant epidermal structures that have autofluorescence under UV or other wavelengths.     

二色补血草的耐盐性研究

通过NaCl胁迫发芽试验、盆栽浇灌NaCl盐水试验、不同含盐量土壤的种植试验,研究二色补血草（Limoniumbicolor）3个时期的耐盐能力。结果表明：高浓度NaCl对二色补血草种子的萌发有明显的抑制作用,种子的发芽率与盐浓度之间呈显著的负相关,种子萌发时盐胁迫的适宜值、临界值、极限值分别是0.49%、1.30%、2.11%;盆栽试验结果表明,二色补血草在2.0%以下的NaCl盐水胁迫下没有表现明显的盐害症状,只表现高浓度NaCl胁迫下,生长量低于低浓度处理的,当处理浓度达到2.4%时,多数植株濒临死亡;不同含盐量土壤的种植试验结果表明,在自然条件下,二色补血草成株耐盐临界值为1.7%～1.8%。试验结果说明二色补血草具有较强的耐盐能力,适合在滩涂盐碱地推广种植。     

二色补血草叶片泌盐结构的扫描电镜观察

对二色补血草盐泉结构进行比较研究,发现二色补血草叶下表皮具有花型盐腺,这些盐腺是由基细胞和帽细胞构成,植物体靠盐腺的泌盐孔和基细胞破碎释盐,表明二色补血草具有演化较高级的双重泌盐结构,是二色补血草能在盐碱地生长而不受盐害的主要原因。     

二色补血草试管苗生根及移栽基质研究

以二色补血草为材料,研究了不同浓度激素配比对试管苗生根量及根长的影响,以及生根苗在不同移栽基质中的成活率和生长状况。结果表明,试管苗在MS+NAA 1.0mg/L培养基中的平均根量最多,在MS+IBA 1.0mg/L培养基中平均根长最长;生根苗在泥炭+珍珠岩(1:1)的基质中生长最好。同时进行降低成本的试验。     

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt‐tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty‐six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt‐secretion capacity in crops.     

二色补血草地上部分的化学成分

二色补血草﹝Limonium bicolor ( Bunge) Kuntze﹞为补血草属( Limonium Mill.)越年生草本植物,耐盐碱和抗逆性强,全草均可入药,具有补血止血、调经益气、抗菌消炎和温中健脾的作用[1],含有黄酮、多糖、挥发油和甾体等成分[2-7],其中,黄酮类成分具有较强的抗氧化能力,多糖对Hela细胞有明显抑制作用[8-11]。前人对二色补血草化学成分和药理活性已有一定研究,但对其地上部分的化学成分尚缺乏系统分析。为此,作者对二色补血草地上部分乙醇提取物进行了分离和鉴定,为该种的进一步开发利用和药理药效研究提供基础实验数据。     

Study on the Physiological Mechanism of Early Flowering and Low Male Fertility of Limonium bicolor Mutant vrl15

As biennial recretohalophytes, Limonium bicolor plants need 2 years to complete their life cycle. A growth habit mutant Vernalization Requirement Loss 15 (vrl15) was obtained by ion implantation. However, the biological characteristics of the mutant were unclear. In the current study, the related traits of vrl15 and some possible reasons for these traits were examined. Compared with wild type (WT), vrl15 can bolt and flower in approximately four months without vernalization. Moreover, vrl15 needed much less time to bolting and flowering than wild-type L. bicolor under different vernalization treatments. After 20 days’ vernalization, bolting vrl15 plants had 24 rosette leaves and bolting WT had 31 rosette leaves. Moreover, the pollen number per anther, the proportion of active pollen, the seed setting rate and the 1000 seed weight of vrl15 were all lower than those of WT. The soluble sugar content and soluble protein content in leaves of the vrl15 were much higher than those of WT sowed at the same time. In addition, the GA content in the leaves of bolting vrl15 was higher than that of the non-bolting WT sowed at the same time and non-bolting vrl15, whereas the contents of ABA and BR were much lower than that of the non-bolting WT. These results indicate that to some extent the increase of GA and decrease of ABA and BR content may be involved in the growth habit and male fertility alteration of mutant vrl15 of L. bicolor.

     

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD₆₀₀) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.     

二色补血草LbGRP基因的克隆及抗逆能力分析

富含甘氨酸RNA结合蛋白(Glycine-rich RNA-binding proteins, GRP)是植物中重要的转录后调控蛋白, 在植物的生长发育及对胁迫的抗性调控等过程中起重要作用。文章从二色补血草(Limonium bicolor (Bunge) O.) cDNA文库中克隆出了富含甘氨酸RNA结合蛋白基因(LbGRP)的全长cDNA序列(GenBank登录号: GQ398238)。为了研究LbGRP的抗逆功能, 将其构建到酵母表达载体pYES2中, 转化到酿酒酵母INVSc1菌株中, 获得重组酵母INVSc1(pYES2-LbGRP), 同时将转空pYES2质粒的酵母INVSc1(pYES2)作为对照。对重组酵母INVSc1 (pYES2-LbGRP)和对照INVSc1(pYES2)进行NaCl、KCl、NaHCO₃、Na₂CO₃、干旱和冷冻胁迫, 比较它们在不同胁迫下的存活率。结果表明, INVSc1(pYES2-LbGRP)在各种胁迫下的存活率明显高于对照INVSc1(pYES2), 证明LbGRP基因具有抗NaCl、KCl、NaHCO₃、Na₂CO₃、干旱和冷冻等胁迫的能力, 推测该基因参与了二色补血草的抗逆调控过程。     

Ion secretion by salt glands of desert iguanas (Dipsosaurus dorsalis)

Unlike the NaCl-secreting salt glands of many birds and reptiles, the nasal salt glands of lizards can secrete potassium as well as sodium, with either chloride or bicarbonate as the accompanying anion. The factors responsible for initiating secretion by the gland and the rates of cation and anion secretion were studied in the desert iguana, Dipsosaurus dorsalis. Lizards were given combinations of ions for several days, and secreted salt was collected daily and analyzed for sodium, potassium, chloride, and bicarbonate. Maximum total cation secretion rate was 4.4+/-0.38 micromol/g/d. Cation secretion ranged from 24% to 100% potassium; even high NaCl loads did not abolish potassium secretion. Maximum bicarbonate secretion was about 0.5 micromol/g/d; chloride was the predominant anion. Secretion rate increased only in response to those treatments that included potassium and/or chloride; sodium ions and other osmotic loads (e.g., sucrose) did not increase secretion. This is in contrast to birds and some other reptiles with salt glands, which initiate NaCl secretion in response to any osmotic load. The specificity of the response of the salt gland of Dipsosaurus may be related to the ecological importance of dietary potassium and chloride for herbivorous desert lizards.     

二色补血草硫氧还蛋白(Trx)的克隆和表达分析

从二色补血草cDNA文库中分离出1个硫氧还蛋白基因全长cDNA序列。基因全长1138bp,其中,5’非翻译（UTR）区128bp,3＇非翻译区212bp,开放阅读框（ORF）全长798bp,编码265个氨基酸,编码蛋白的分子量为28．58kDa,理论等电点（pI）为9．68。BlastP分析表明二色补血草Trx与拟南芥Trx序列同源性为52％,与葡萄7h序列同源性为76％,从11个物种的氨基酸多序列比对可以看出Trx氨基酸序列保守性较高。实时定量RT-PCR方法检测低温、NaCl和PEG胁迫不同时间后的基因在二色补血草中表达模式的结果表明,NaCl能诱导Trx基因在二色补血草叶中表达,胁迫24h后达到高峰,而聚乙二醇和低温处理则抑制Trx在二色补血草根和叶的表达。     

一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析

从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。     

二色补血草LbDREB基因在酵母中的抗逆性分析

利用酵母表达系统研究了二色补血草的DREB基因（LbDREB）对不同胁迫的抗性。将LbDREB构建到酵母表达载体pYES2中,转化到酿酒酵母INVSc1菌株中,并以转空pYES2质粒的酵母INVSc1（pYES2）作为对照,通过比较两种酵母在不同胁迫下的存活率来研究LbDREB基因对NaCl、KH_2PO_4、Na_2CO_3、NaHCO_3、低温、干旱、CuSO_4和CdCl_2胁迫的抗性。结果表明,LbDREB转化的酵母在各种胁迫下的存活率均明显高于转空pYES2的对照酵母,说明LbDREB基因除了具有传统认为的抗旱、耐盐、抗寒的作用外,还具有抗KH_2PO_4、Na_2CO_3、NaHCO_3、CuSO_4和CdCl_2等胁迫的能力。