Immunofluorescent Localization of a Connexin 26-like Protein at the Surface of Mesophyll Protoplasts from Vicia faba L. and Helianthus annuus L.

Mesophyll protoplasts from three week old leaves of Helianthus annuus L. and from four week old leaves of Vicia faba L. were incubated with polyclonal, monospecific antibodies, raised against either cx 32 or cx 26 mouse liver connexin. Crossreactions were visualized by FITC-labeled anti-rabbit antibodies. Incubations with the cx 26 antibody resulted in fluorescing spots on protoplast surfaces of both plant species, indicating the presence of a polypeptide, immunologically related to the animal cx 26. A plant protein, exhibiting similarities to cx 26, would present a new member of connexin-like plant proteins. Controls, performed with preimmune serum or with the FITC-conjugate alone, were negative. Immunofluorescing spots were not obtained after incubations with the cx 32 antibody. Since the existence of a cx 32-like plant protein, associated with ultrastructures of plasmodesmata and the plasma membrane, is meanwhile established, several explanations for the failed attempt to demonstrate a cx 32 antibody labeling at protoplast surfaces are discussed.     

Tissue- and Cell-Specific Distribution of Connexin 32-and Connexin 26-related Proteins from Vicia faba L.

Different tissues (leaf lamina and midveins, epidermis, primary roots, etiolated shoots, hypocotyl, petioles) and protoplasts from mesophyll, guard cells, and petioles of Vicia faba L., obtained from defined developmental stages were analyzed with polyclonal antibodies raised against mouse liver connexins 26 and 32. Immunostaining after treatment with CX 26 antibodies showed two polypeptides of 21 and 16 kD in all tissues examined except for hypocotyl and epidermis. Additionally, a stronger immunoresponse at 40 kD occurred in extracts from leaf material, mesophyll protoplasts, and roots. Incubation with the CX 32 antibodies resulted in an immunoreactive band at 29 kD in mesophyll protoplast samples, and, a very weak band in extracts from leaf material. A 36 kD polypeptide, present in samples prepared from root, etiolated shoots, petioles, and midveins, did not appear in leaf laminas and mesophyll protoplasts. Extracts from guard cells did not show any immunoreactive band. The tissue- and cell-specific distribution of CX 23- and CX 26-related proteins is supposed to reflect diversities in physiological functions together with alterations in plasmodesmatal ultrastructures during development.     

Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

The regulation of stomatal movement is one of the most important signaling networks in plants.The H -ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H -ATPase inhibitor,vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Viciafaba L., but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous findings that H -ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases),we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase.As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V. faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.     

Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

The regulation of stomatal movement is one of the most important signaling networks in plants.The H^ -ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H^ -ATPase inhibitor,vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Vicia faba L., but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous findings that H~-ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases),we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase,As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V.faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.     

H2S介导ABA诱导蚕豆气孔运动的生理机制研究

以蚕豆为实验材料,利用药理学实验和分光光度法,研究了ABA处理及ABA与H2S合成抑制剂共处理对蚕豆气孔运动的影响,以及体内H2S水平、H2S合成酶L-/D-半胱氨酸脱巯基酶(磷酸吡哆盐依赖性酶)活性变化.结果表明:(1)光下H2S的合成抑制剂羧甲氧基胺半盐酸盐(AOA)、羟氨(NH2OH)、L-/D-半胱氨酸脱巯基酶分解产物C3H3KO3+NH3均明显抑制ABA诱导的蚕豆气孔关闭;(2)外源ABA能够明显提高叶片的H2S水平及L-/D-半胱氨酸脱巯基酶活性;(3)AOA、NH2OH、C3H3KO3和NH3均可以逆转ABA所引起的H2S水平及L-/D-半胱氨酸脱巯基酶活性的升高.研究发现,ABA可通过增强L-/D-半胱氨酸脱巯基酶活性,促进L-/D-半胱氨酸分解生成H2S,进而诱导蚕豆气孔关闭.     

蚕豆水孔蛋白可能参与微丝骨架对气孔运动的调节

水孔蛋白的抑制剂HgCl2可明显抑制壳梭孢菌素（FC）和微丝骨架的解聚剂细胞松弛素D（CD）对蚕豆保卫细胞原生质体膨胀的诱导作用,而对微丝骨架的稳定剂鬼笔环肽（phalloidin）的抑制作用影响不明显。这表明水孔蛋白可能介导了FC和微丝骨架对气孔运动的调节。     

二氧化硫对蚕豆叶片伤害类型的研究

二氧化硫熏气处理后对蚕豆（ＶｉｃｉａｆａｂａＬ．）叶片的伤害可划为３种类型,即不可见伤害、可逆的可见伤害以及不可逆伤害。采用动态熏气的方法初步估计了３类伤害的阈值,不可见伤害阈值小于１３１．２μｇ／ｍ￣３×８ｈｓ,可见伤害阈值为１３２２．８μｇ／ｍ￣３×２ｈｓ,１３２２．８μｇ／ｍ￣３ＳＯ＿２处理时不可逆伤害阈值介于１６ｈｓ（累积小时数,下同）和２４ｈｓ之间,而以７７５．０μｇ／ｍ￣３ＳＯ＿２处理时,则介于３２ｈｓ和４０ｈｓ之间。认为超氧化物歧化酶活性作为不可见伤害和不可逆伤害的指标较为适宜,可见伤害阈值则可沿用传统的５％伤害叶面积作指标。     

Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields

Growth, mitotic index, and growth rate recovery were determined for Vicia faba L. roots exposed to 60-Hz electric fields of 200, 290, and 360 V/m in an aqueous inorganic nutrient medium (conductivity 0.07-0.09 S/m). Root growth rate decreased in proportion to the increasing strength; the electric field threshold for a growth rate effect was about 230 V/m. The induced transmembrane potential at the threshold exposure was about 4-7 mV. The mitotic index was not affected by an electric field exposure sufficient to reduce root growth rate to about 35% of control. Root growth rate recovery from 31-96% of control occurred in 4 days after cessation of the 360 V/m exposure. The results support the postulate that the site of action of the applied electric fields is the cell membrane.     

In Vitro and in Vivo Phosphorylation of Stomatal Phosphoenolpyruvate Carboxylase from Vicia faba L.

The in vitro and in vivo phosphorylation of stomatal phosphoenolpyruvate carboxylase [EC 4.1. 1.31] from Vicia faba L. was demonstrated by feeding the concentrated enzyme after 0–70% ammonium sulfate precipitation of guard cell protoplasts with ³²P and subsequent analysis of autoradiograms and Western immunoblots, after SDS-PAGE, of protein samples. The in vitro and in vivo results provide evidence for a radioactive labeling of the two stomatal PEPCase bands (112 and 110 kDa).     

酪氨酸蛋白磷酸酶(PTPase)参与水杨酸调控蚕豆气孔运动的信号转导

研究酪氨酸蛋白磷酸酶(PTPase)的抑制剂氧化苯胂(PAO)、钒酸钠(NaVO3)和Zn2 对水杨酸(SA)调控蚕豆气孔运动影响的结果表明,0~1mmol·L-1 PAO、0~4mmol·L-1 NaVO3和0~4mmol·L-1Zn2 对光诱导蚕豆气孔开度变化的影响不大,但都可以抑制黑暗或SA诱导的气孔关闭,据此推测,PTPase可能参与SA诱导气孔关闭的信号转导过程。     

A combination of thidiazuron and benzyladenine promotes multiple shoot production from cotyledonary node explants of faba bean (Vicia faba L.)

A procedure for multiple shoot formation from cotyledonary node explants of faba bean (Vicia faba L.cv.S.Ghdar) cultured on MS medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced a higher number of shoots than with either cytokinin alone. The highest number of shoots was obtained when explants from 7-day-old seedlings were cultured on MS medium supplemented with TDZ and BA (2 mgl–1 each) for 31 days before transfer to hormone-free MS medium for elongation. Shoots produced in vitro were rooted on half-strength agar-solidified MS basal medium or with 0.25 or 0.5 mgl–1 naphthalenacetic acid (NAA) prior to transfer to green house conditions. This procedure was found to be applicable to seven other cultivars of faba bean from widely diverse provenances. Thus, it can be advantageously applied to the production of transgenic faba bean plants.     

He-Ne激光处理不同时期蚕豆幼苗对抗氧化系统的影响

采用功率为3.50 mW/mm2的He-Ne激光处理不同时期蚕豆,研究其四叶期叶片中丙二醛(MDA)含量,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性及同工酶谱的变化。结果表明,与对照相比,He-Ne激光处理不同时期蚕豆,MDA含量均显著降低,且各处理间没有显著差异;SOD、POD、CAT酶活性有不同程度提高。SOD、POD同工酶的酶谱在浸种24小时和胚芽露头期经激光处理后改变,在一叶期处理后没有改变;CAT同工酶谱没有变化;在一叶期处理的三种同工酶谱都没有改变。     

Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of Vicia faba L.

Plasma membranes from three week old leaves of Vicia faba L. were enriched by aqueous two-phase partitioning to high purity. Plasma membrane proteins were immunoblotted with polyclonal, monospecific antibodies raised against mouse liver connexins (cx) 32 and 26. Immunostaining after treatments with cx 32 antibodies revealed the existence of a 29 kDa protein, clearly enriched in the plasma membrane fraction. An additional immunoreactive band of 20 kDa, possibly a degradation product of the 29 kDa protein, was found in the soluble fraction. When immunoblots were incubated with cx 26 antibodies, a 40 kDa band with a strong immunoresponse appeared, assumed to present the dimeric form of a 21 kDa, cx 26-like plant protein. The monomeric form could be only obtained when intact leaf material or mesophyll protoplasts from three week old plants were directly SDS-extracted. Furthermore, in young, one week old leaves, the monomer seems to exist in larger amounts, together with another crossreacting 35 kDa protein. The 29 kDa (cx 32-related) as well as the 40 kDa (cx 26-related) polypeptide is obviously located in the plasma membrane. The 40 kDa protein has to be considered as a new connexin-like plant protein.     

我国蚕豆根瘤菌的数值分类及其16SrDNA PCR-RFLP研究

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用MINTS软件进行聚类分析。结果表明,全部供试菌株在59％的相似水平上聚在一起,在80％的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR—RFLP分析,在85％相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

我国蚕豆根瘤菌的数值分类及其16S rDNA PCR-RFLP研究*

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用M INTS软件进行聚类分析。结果表明,全部供试菌株在59%的相似水平上聚在一起,在80%的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR-RFLP分析,在85%相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

繁殖群体量及隔离对蚕豆种质遗传完整性的影响

为研究繁殖群体量和隔离方式对常异花授粉作物蚕豆种质繁殖更新的影响,以9份蚕豆地方种质为对象,以国家库保存的原种为对照群体,采用AFLP分子标记方法,对比了50株和20株群体量及开放无隔离群体和网棚隔离群体更新后代的遗传完整性差异。结果表明:与对照群体相比,50株和20株群体的多态性位点数、多态位点百分率、每位点有效等位基因数、香农指数、遗传多样性指数均出现不同程度下降,但下降幅度20株大于50株群体;遗传相似性和UPGMA聚类分析表明50株群体与对照群体的遗传相似性高于20株群体;网棚隔离可降低群体间串粉与花粉污染,但其遗传完整性却较开放无隔离群体低。     

水杨酸、茉莉酸和乙烯在调控蚕豆气L运动中的相互关系

以蚕豆下表皮为材料研究了水杨酸、(±)茉莉酸和乙烯对气孔运动的影响及其相互关系。结果表明,在一定范围内,水杨酸和乙烯利都可诱导气孔关闭,并且二者能够相互增强其作用强度;(±)茉莉酸能够促进气孔开度增大,加入(±)茉莉酸减弱了乙烯利对气孔运动的影响,(±)茉莉酸和乙烯利存在拮抗效应。降低内源乙烯的水平可以增强(±)茉莉酸促进气孔张开的作用、降低水杨酸的诱导气孔关闭效应。而水杨酸和(±)茉莉酸之间的关系比较复杂。     

Ca~(2+)参与NO对蚕豆气孔运动的调控

观察了Ca2 + 、Ca2 + 的螯合剂和Ca2 + 通道抑制剂对NO调控的蚕豆气孔运动的影响。结果表明 ,NO的供体 1～ 10 0 μmol/LSNP (sodiumnitroprusside ,硝普纳 )可诱导气孔关闭 ;除去表皮条缓冲液中的Ca2 + 后 ,NO不再影响气孔的运动 ;Ca2 + 的螯合剂EGTA和BAPTA几乎可以完全抑制NO诱导的气孔关闭作用 ;胞内钙通道抑制剂钌红 (rutheniumred)和L型Ca2 + 通道阻断剂硝苯吡啶 (nifedipine)能够减弱SNP诱导气孔运动的关闭趋势 ;加入Ca2 + 通道抑制剂LaCl3 ,则外源NO失去其诱导气孔关闭的作用。说明在NO调控的气孔运动中 ,在NO信号途径的下游可能涉及来自胞内和胞外Ca2 + 的参与 ,并且胞外Ca2 + 更为重要。     

Canopy development of a determinate and an indeterminate cultivar of Vicia faba L. under contrasting plant distributions and densities

Field experiments were conducted in 1987 and 1988 to quantify differences in canopy formation between an indeterminate and a determinate genotype of Vicia faba L., grown at two plant densities and three spatial distributions. The number of stems per unit area produced by determinate plants was related to the growth rate before flowering. Leaf production per stem per unit of thermal time was similar in both plant types, but twice as many leaves per stem were produced by the indeterminate cultivar. The indeterminate cultivar produced fewer and smaller leaves in the warmer and drier weather of 1988 than in 1987. The determinate genotype produced similar sizes and numbers of leaves in both years, but fewer tillers developed in 1988 than in 1987. Accordingly, leaf mass per unit ground area was greater in 1987 than in 1988 in both genotypes. Except during early flowering, relationships between leaf mass and leaf area were constant, with higher specific leaf areas in the determinate than the indeterminate genotype. Shoot dry matter partitioning into leaves was identical in both years for indeterminate plants, but differed in determinate ones.
It is concluded that canopy development is regulated through individual leaf weight and leaf number per stem in non-tillering indeterminate, and by stem numbers per unit area in tillering determinate plants.     

Influence of Ca2+ and pH on the Stability of Different Boron Fractions in Intact Roots of Vicia faba L.

Abstract: An experimental setup was developed which allowed the detection of different boron fractions in intact plant roots. The B fractions were water-soluble B (WSB), BaCI₂-soluble B (BaSB) and the respective insoluble fractions (WIB and BaIB). In root tips of V. faba seedlings, WSB represents approx. 4%, BaSB 9% of B_tot. BaSB consists of (at least) two compounds, the larger of which (65 % ) interacts with phosphate and is considered a soluble B complex. Within 2 h of B deficiency, WSB and a large part of BaSB disappear, suggesting that they may be involved in early deficiency reactions in root tips. Concerning insoluble fractions, there was no decline in WIB within the first 6 h of B deficiency. Within the same time frame, BaIB dropped to 35% of the initial B content, indicating a stabilizing effect of Ca on B complexes under in vivo conditions. The concentration of WSB in root tips was lower at pH 3.6 as compared to pH 6.0, indicating that changes in apoplastic pH rapidly alter the equilibrium between free and bound B. WIB was more tightly bound in older root parts, as indicated by a maximum loss of 34% of B_tot after 24 h of continuous B removal, as compared to 99.5% in root tips. Our results indicate that different fractions of B (additional to B-RGII) are affected in the early stages of B deficiency and that Ca is not only a stabilizing agent for B complexes but is also necessary to bind soluble B complexes in the apoplast of intact roots.