蚕豆（Vicia faba L．）叶肉细胞中ABA的胶体金免疫电镜定位

利用胶体金免疫电镜定位技术对蚕豆叶肉细胞中ＡＢＡ定位的研究表明,在以ＡＢＡ抗体处理的切片中,叶绿体有大量的金颗粒标记,细胞质和细胞核也有金颗粒标记,但液泡和细胞壁中没有金颗粒标记。免疫染色前用胰蛋白酶处理可显著增强金颗粒标记密度,而不用ＥＤＣ固定或以免疫前兔血清处理的切片中几乎没有金颗粒标记。本实验为蚕豆叶肉细胞中ＡＢＡ的分布提供了直接的证据并说明了该技术是研究ＡＢＡ定位的一种可靠的方法。     

生物素标记钙调素用于植物钙调素结合蛋白的检测

用生物素标了己了花椰菜ＣａＭ。生物素标记的ＣａＭ具有与天然ＣａＭ相似的Ｃａ２＋依赖电泳特性,可激活ＣａＭ依赖性磷酸二酯酶,能够检测出５０ｎｇ的磷酸二酯酶。利用它建立了检测植物ＣａＭ结合蛋白的生物素－覆盖法（Ｂｉｏｔｉｎ－ｏｖｅｒｌａｙ）并证实酶标亲和素可与胡萝卜愈伤组织内６４ｋＤ蛋白质非特异结合,因此将此法运用于植物材料时必需设置酶标亲和素处理的对照。用生物素－覆盖法检测胡萝卜愈伤组织形成过程中的ＣａＭ结合蛋白时可检出２,４－Ｄ诱导的ＣａＭ结合蛋白。     

细胞松弛素B对蚕豆气孔运动的影响

以蚕豆叶片下表皮条为材料,研究了微丝在气孔运动中的作用。利用肌动蛋白纤丝专一性抑制剂──细胞松弛素Ｂ（ＣＢ）预处理后,再用诱导气孔运动的因子处理表皮条,在显微镜下观测气孔孔径的变化。结果显示,用ＣＢ处理开放或关闭状态气孔,其开度均不发生变化;ＣＢ处理使微丝解聚,气孔运动被抑制;且ＣＢ处理后气孔的运动是可以恢复的。实验进一步表明,开放气孔经１０ｍｇ／Ｌ的ＣＢ预处理后,ＡＢＡ、Ｃａ２＋及暗诱导气孔关闭的作用均不同程度地受到抑制,推测微丝可能参与ＡＢＡ、Ｃａ２＋及暗诱导的气孔关闭过程;关闭气孔经１０ｍｇ／Ｌ的ＣＢ预处理后,Ｋ＋和（或）光诱导气孔开放的作用受到抑制,推测微丝可能参与光及Ｋ＋诱导的气孔开放过程。     

基于生态系统评价的山水林田湖草生态保护与修复体系构建研究——以乌梁素海流域为例

山水林田湖草系统保护与修复工程实施的重要目标是维护和提升区域生态系统服务。从乌梁素海流域山水林田湖草的生态现状与功能出发,对乌梁素海流域水土流失、土地沙化等生态敏感性及土壤保持、水源涵养、生物多样性等生态功能重要性进行系统评价,形成基于生态敏感性和生态功能重要性相结合的空间格局评价结果。以维护和提升人类福祉所需的重要生态系统服务为目标,以乌梁素海流域生态敏感性和生态功能重要性相结合的空间格局评价结果为基础,制定了“一中心、二重点、六要素、七工程”的乌梁素海山水林田湖草生态保护与修复体系,并基于此将乌梁素海流域生态保护修复分为6个主要治理区域,形成“四区、一带、一网”的生态安全格局,通过具体工程实施,流域生态环境质量和生态服务能力将取得明显提升,防风固沙能力有效增强,生物多样性持续改善,水环境质量稳定达标,生态系统的稳定性明显加强。通过乌梁素海流域的分析案例为流域山水林田湖草生态保护与修复关键区域的识别提供了定量分析方法,为流域尺度构建生态安全格局、实现山水林田湖草系统保护和修复提供思路和途径。     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0715-0) contains supplementary material, which is available to authorized users.     

Regulation of stomatal opening by the guard cell expansin AtEXPA1

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.Key words: expansin, stomatal movement, AtEXPA1, guard cell, wall looseningStomatal movement is the most popular model system for cellular signaling transduction research. A complicated complex containing many proteins has been proposed to control stomatal responses to outside stimuli. The known regulation factors are primarily located in the nucleus, cytoplasm, plasma membrane and other intracellular organelles.^1,2 Although the cell wall structure of the stomata is different from that of other cells,^3,4 the presence of stomatal movement regulation factors in the cell wall has seldom been reported in reference ⁵. In our previous work, we found that extracellular calmodulin stimulates a cascade of intracellular signaling events to regulate stomatal movement.⁶ The involvement of this signaling pathway is the first evidence that cell wall proteins play an important role in regulation of stomatal opening. Cell wall-modifying factors constitute a major portion of cell wall proteins. However, the role of these factors in the regulation of stomatal movement is not yet known.Expansins are nonenzymatic proteins that participate in cell wall loosening.^7–9 Expansins were first identified as “acid-growth” factors because they have much higher activities at acidic pHs.^10,11 It has been reported that expansins play important roles in plant cell growth, fruit softening, root hair emergence and other developmental processes in which cell wall loosening is involved.^7–9,12,13 Wall loosening is an essential step in guard cell swelling and the role of stomatal expansins was investigated. AtEXPA1 is an Arabidopsis guard-cell-specific expansin.^13,14 Over-expressing AtEXPA1 increases the rate of light-induced stomatal opening,^14,15 while a potential inhibitor of expansin activity, AtEXPA1 antibody, reduces the sensitivity of stomata to stimuli.¹⁴ We showed that the transpiration rate and the photosynthesis rate in plant lines overexpressing AtEXPA1 were nearly two times the rates for wild-type plants (Fig. 1). These in plant data revealed that expansins accelerated stomatal opening under normal physiological conditions. In addition, the increases in the transpiration and photosynthesis rates strongly suggested the possibility of exploiting expansin-regulated stomatal sensitivity to modify plant drought tolerance. Compared with the effect of hydrolytic cell wall enzymes, the destruction of cell wall structures induced by expansins is minimal. In addition, it is very difficult to directly observe the changes in the guard cell wall structure caused by expansins during stomatal movement. Our recent work showed that, in AtEXPA1-overexpressing plants, the volumetric elastic modulus is lower than in wild-type plants,¹⁴ which indicates the wall structure was loosened and that the cell wall was easier to extend. Taken together, our data suggest that expansins participate in the regulation of stomatal movement by modifying the cell walls of guard cells.Open in a separate windowFigure 1Effects of AtEXPA1 overexpression on transpiration rates and photosynthesis rates. The transpiration rate (left) and photosynthesis rate (right) of wild-type and transgenic AtEXPA1 lines were measured at 10:00 AM in the greenhouse after being watered overnight. The illumination intensity was 180 µmol/m²·s. Bars represent the standard error of the mean of at least five plants per line.It is well known that the activation of proton-pumping ATPase (H⁺-ATPase) in the plasma membrane is an early and essential step in stomatal opening.¹⁶ The action of the pump results in an accumulation of H⁺ outside of the cell, increases the inside-negative electrical potential across the plasma membrane and drives potassium uptake through the voltage-gated, inward-rectifying K⁺ channels.^17–19 The main function of the H⁺ pump is well accepted to create an electrochemical gradient across the plasma membrane; however, the other result is the acidification of the guard cell wall, which may also contribute to stomatal opening. A possible mechanism responsible for this effect is as follows. Expansins are in an inactive state when the stomata are in the resting state. Stomatal opening signals induce wall acidification and activate expansins. Then, the expansins move along with cellulose microfibrils and transiently break down hydrogen bonding between hemicellulose and the surface of cellulose microfibrils,^20,21 facilitating the slippage of cell wall polymers under increasing guard cell turgor pressure. The guard cell then swells and the stomata open (Fig. 2).Open in a separate windowFigure 2Model of how guard cell wall expansins regulate stomatal opening. Environmental stimuli, e.g., light, activate guard cell plasma membrane H⁺-ATPases to pump H⁺ into the extracellular wall space. The accumulation H⁺ acidifies the cell wall and induces the activation of expansin. The active expansin disrupts non-covalent bonding between cellulose microfibrils and matrix glucans to enable the slippage of the cell wall. The wall is loosened coincident with guard cell swelling and without substantial breakdown of the structure.Although our results indicate that AtEXPA1 regulates stomatal movement, the biochemical and structural mechanism by which AtEXPA1 loosens the cell wall remains to be discovered. It remains to figure out the existing of other expansins or coordinators involving in this process. In addition, determining the roles of expansins and the guard cell wall in stomatal closing is another main goal of future research.     

植物水孔蛋白的亚细胞分布与生理功能研究浅析

水孔蛋白(aquaporin,AQP)因具有水转运活性而得名,然而随着研究的深入,水孔蛋白转运活性的多样性与生理功能的多样性不断被报道．本文综合分析了植物水孔蛋白亚细胞定位与功能多样性的研究进展,重点综述了植物水孔蛋白广泛的亚细胞分布特点,以及亚细胞上的再分布现象与植物水孔蛋白生理功能多样性间的关系,并对植物水孔蛋白研究中存在的 问题及研究方向进行了分析,认为水孔蛋白多样化的生理功能的作用机制需要结合其组织定位与亚细胞定位进行分析才能 揭示．     

水稻 Ca2+/H+ 反向转运体 OsCAX3 的功能分析和亚细胞定位研究

Ca2+/H+ 反向转运体作为一类 Ca2+外向转运器,在植物的营养和信号转导中起着非常重要的作用 . 克隆了水稻 Ca2+/H+ 反向转运体基因 OsCAX3 ,序列分析表明 OsCAX3 具有 11 个跨膜区,其中在第 6 和第 7 个跨膜区之间有一个 17 个氨基酸组成的酸性基序 (acid motif) ,功能互补实验证明 OsCAX3 具有转运 Ca2+ 的功能,并且其 N 端 26 个氨基酸序列对转运 Ca2+ 具有一定的抑制作用 . RT-PCR 分析表明 OsCAX3 的表达受到外源 Ca2+ 的诱导 . 利用 PSORT prediction 进行亚细胞定位分析,和利用 OsCAX3-GFP 融合蛋白瞬时表达分析证明, OsCAX3 定位于细胞质膜 . 以上结果表明, OsCAX3 是一种定位于细胞质膜上的 Ca2+/H+ 反向转运体 .