Characterization and temperature regulation of soluble proteins of a male sterile tomato mutant

The soluble proteins of the normal and male-sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum) grown in different temperatures were analyzed by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The normal and mutant stamens had some common proteins, but certain proteins were either present or more enriched in one genotype than in the other. The other floral organs of the normal and mutant showed no major differences in proteins, suggesting that the sl-2/sl-2 allele is active primarily in anther development. Normal and mutant stamens grown in high temperatures were enriched in some proteins in comparison to the intermediate temperatures. At low temperatures, the protein pattern of normal and mutant stamens was essentially similar.     

Protein analysis during the ontogeny of normal and male sterile stamenless-2 mutant stamens of tomato (Lycopersicon esculentum Mill.)

The levels and synthesis of proteins during the ontogeny of normal and male sterile stamenless-2 (sl-2/sl-2) mutant stamens of tomato (Lycopersicon esculentum) were examined. The mutant stamens contained low levels of soluble protein which were related to reduction in protein synthesis. The mutant stamens, however, possessed many polypeptides similar to the normal and synthesized a 53-kd polypeptide at stages when there are abnormalities in tapetum development. The mutant stamens also possessed a 23-kd and some low molecular weight polypeptides that were considered as degradative proteins. Normal stamens exhibited the synthesis of many polypeptides not found in the mutant, from microspore mother cell to the preanthesis stages. In addition, at the time of pollen maturation there was a greater synthesis of several polypeptides, particularly those of 42 and 37 kd. Although the causative mechanisms of male sterility in the sl-2/sl-2 mutant are not known, the synthesis, and the lack, of specific polypeptides reported here appears to be associated with pollen degeneration.This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to V.K.S.     

Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile 'stamenless-2' mutant of tomato (Lycopersicon esculentum Mill.)

The indole-3-acetic acid (IAA) concentration in leaves and stamens of the normal and a temperature-sensitive male sterile ‘stamenless-2′ (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.), grown under three temperature conditions, was measured by gas chromatography — mass spectrometry — selected ion monitoring (GC-MS-SIM) and by enzyme-linked immunosorbant assay (ELISA). At low (LTR, 18°C day/15°C night), intermediate (ITR, 23°C day/18°C night), and high temperatures (HTR, 28°C day/23°C night), the mutant leaves had approximately 10 to 20 times higher IAA concentrations, respectively, than the normal leaves under these temperature regimes. Similarly, the stamens of mutant flowers had approximately five and eight times higher IAA concentration at ITR and HTR, respectively, than the normal flowers. In the low temperature reverted mutant stamens, however, the level of IAA was similar to that in normal stamens. Also, with an increase in temperature, there was an increase in the level of IAA in the leaves and stamens of mutant plants. However, different temperatures had no appreciable effect on the IAA content of leaves and stamens of normal plants. It is suggested that the high IAA content in leaves and stamens of the stamenless-2 mutant is one of the factors associated with male sterility and carpellization of stamens in this mutant.     

Abscisic acid in a male sterile tomato mutant and its regulation by low temperature

The role of abscisic acid in male sterility in the stamenless21 (sl-2) mutant of tomato (Lycopersicon esculentum) was investigated. Vegetative and floral parts (except pistil) of sl-2 contain greater amount of abscisic acid (ABA) than the normal wild type. The maximum difference in ABA content between sl-2 and normal tissues was in stamens, and the increase in ABA level in sl-2 stamens coincided with first signs of abnormalities in the anthers. Low temperatures restore male fertility in sl-2 and there was a concomitant drop in ABA level in sl-2 leaves and stamens. These observations, along with our earlier reports, suggest that male sterility in sl-2 is a manifestation of hormonal imbalance involving high ABA, and that low temperature regulation of male sterility is mediated through reduction in ABA content.Key words: Abscisic acid, Lycopersicon esculentum, male sterility, temperature, tomato.      

Activity and isoenzyme composition of peroxidase and esterase in fertile and male-sterile lines of tomatoes (Lycopersicon esculentum Mill.)

A comparative study of the isoenzyme patterns of esterase and peroxidase and overall peroxidase activity in stamens of male-sterile (MS) lines of Pearson ms-35 and P ms-35aa and of the respective male-fertile (MF) tomato plants have been conducted. The study has been made at two stages of stamens development — tetrad and pollen. Higher activities of the esterase isoenzymes in the MF stamens than that of MS in both ontogeny stages have been found. The slow moving esterase isoenzymes both of the MF and the MS stamens are the major isoenzymes in the early stage and are connected with tapetum development while the fast moving esterase isoenzymes are connected with pollen formation in the later ontogeny stage. Overall peroxidase levels in the MS stamens were higher than those of MF. The peroxidase patterns of the MS lines are also characterized by the greater number of isoenzymes and also the presence of specific isoenzymes, the contrast between the MF and the MS stamens being more strongly expressed at the later stage of development. A strong similitude between esterase and peroxidase patterns behaviour in both MS lines has been found.     

Amylolytic Activity and Carbohydrate Levels During the Stamen Ontogeny of a Male Fertile, and a 'Gibberellin-Sensitive' Male Sterile Mutant of Tomato (Lycopersicon esculentum)

The activity of amylases, and the level of starch and solublesugar content were analysed in the normal (+/+), a ‘gibberellin-sensitive’male sterile stamenless-2 (sl-2/sl-2) mutant, and GA³-revertedsl-2/sl-2 mutant stamens of tomato (Lycopersicon esculentumMill.), at various stages of development. In the mutant stamens,amylolytic activity did not differ from that of the normal untilthe tetrad stage, but thereafter it was significantly lowerthan that of normal stamens. The starch content also did notdiffer in the two lines at early stages of development. However,at later stages it decreased in normal stamens, whereas it remainedunchanged in the mutant. The soluble sugar content graduallyincreased during the development of normal stamens. But in mutantstamens, it remained the same throughout development and wassignificantly lower than the normal. In GA₃-reverted mutantstamens, the amylolytic activity and the level of starch andsoluble sugars were comparable to normal stamens. It is proposedthat the sl-2/sl-2 mutation, through its effects on endogenousgibberellins, affects the activity of amylases which, in turn,result in lower sugar levels leading ultimately to abnormalpollen development. Key words: Amylases, male-sterility, tomato     

Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) : I. Level of Polyamines and Their Biosynthesis in Normal and Mutant Flowers

The levels of free putrescine, spermidine, and spermine, and the activities of ornithine decarboxylase and s-adenosylmethionine decarboxylase were determined in the floral organs of the normal and a male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.). Under the intermediate temperature regime, all mutant floral organs possessed significantly higher levels of polyamines and enzyme activities than their normal counterparts. In the low temperature-reverted mutant stamens, the polyamine levels and the activity of PA biosynthetic enzymes were not significantly different from the normal. It is suggested that the abnormal stamen development in the sl-2/sl-2 mutant is, in part, related to elevated levels of endogenous PAs.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

FLOWER CULTURE OF A MALE STERILE STAMENLESS-2 MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM)

Young floral buds of a male sterile stamenless-2 (sl-2/sl-2) mutant of tomato were cultured, at the sepal primordia stage, in a liquid Murashige and Skoog medium containing either benzylaminopurine (BAP) or gibberellic acid (GA₃) or both. In the basal medium (BM), the buds initiated petal and stamen primordia only and they showed limited development. In buds grown in BM supplemented with 10^–6 M BAP, all types of organ primordia were initiated but the petals remained small and the stamens and carpels were immature. Well-developed flowers with a normal complement of floral organs were, however, produced in a medium containing both BAP (10^–6 M) and GA₃ (10^–7 M to 10^–5 M). The development of stamens was variable and ranged from the complete absence of microsporogenesis to the formation of abnormal pollen. Gynoecium development was normal and ovules with megaspores were produced in the ovary. The results show that male sterility in the sl-2/sl-2 mutant can be expressed in vitro and that GA₃ is essential for the in vitro growth and development of all the floral organs of this mutant.     

Characterization of anther differentiation in cytoplasmic male sterile maize using a specific isozyme system (esterase)

Summary During anther development, characterized in maize plants with N cytoplasm, certain esterase isozymes in non-microspore cells decrease in amount with anther age and new isozymes appear in the developing microspores. In anthers from male sterile plants with cms T or cms C cytoplasm, neither of these changes in esterase patterns occurred. In anthers from plants with cms S cytoplasm, the decrease in the esterases of non-microsporogenous cells was observed but not the appearance of microspore esterases. In lines carrying cms S cytoplasm and nuclear restorer genes, esterase changes during anther development were as in normal fertile anthers. These results are discussed with respect to the phenomenon of cytoplasmic male sterility in the different maize genotypes.     

Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) : II. Effects of Polyamines and Their Biosynthetic Inhibitors on the Development of Normal and Mutant Floral Buds Cultured in Vitro

The floral organs of the male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.) contain significantly higher level of polyamines than those of the normal (R Rastogi, VK Sawhney [1990] Plant Physiol 93: 439-445). The effects of putrescine, spermidine and spermine, and three different inhibitors of polyamine biosynthesis on the in vitro development of floral buds of the normal and sl-2/sl-2 mutant were studied. The polyamines were inhibitory to the in vitro growth and development of both the normal and mutant floral buds and they induced abnormal stamen development in normal flowers. The inhibitors of polyamine biosynthesis also inhibited the growth and development of floral organs of the two genotypes, but the normal flowers showed greater sensitivity than the mutant. The inhibitors also promoted the formation of normal-looking pollen in stamens of some mutant flowers. The effect of the inhibitors on polyamine levels was not determined. The polyamine-induced abnormal stamen development in the normal, and the inhibitor-induced production of normal-looking pollen in mutant flowers support the suggestion that the elevated polyamine levels contribute to abnormal stamen development in the sl-2/sl-2 mutant of tomato.     

Studies on the Molecular Weights of Esterase and Peroxidase Isozymes of Maize

The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed.     

萝卜贮藏期可溶性蛋白及同工酶研究

试验以４个萝卜（Ｒａｐｈａｎｕｓ ｓａｔｉｖｕｓ Ｌ．）品种为试标,研究了贮藏期可溶性蛋白含量及ＰＡＧＥ蛋白图谱、酯酶和淀粉酶的同工酶谱。试验结果表明,在贮藏期蛋白浓度随贮存时间延长而下降,ＰＡＧＥ蛋白谱带显示不仅有品种之间蛋白种类的差异而且有贮藏期不同阶段的差异,淀粉酶同工酶谱显示随贮藏时间延长,酶活性下降,同工酶谱带增加,酯酶在贮藏过程中酶活性呈现先升高再下降然后再升高的变化,同工酶谱带贮藏中     

自然突变株系——大叶龙茶核型及酯酶同工酶研究

对自然突变株系———大叶龙茶进行了核型研究 ,并对 7个茶叶品种进行了酯酶同工酶研究。大叶龙的核型公式为 2n =2x =3 0 =1 8m +1 0sm(2SAT) +2st(2SAT) ,酯酶同工酶酶谱能反映茶叶品种的区域性特征。大叶龙茶酯酶酶谱发生变异 ,即突变体 (大叶龙 )比母株 (宁洲群体种 )少一条C5酶带。     

Decapitation impacting effect of topically applied chlorpyrifos on acetylcholinesterase and general esterases in susceptible and resistant German cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing alpha-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

离子束介导大豆DNA导入小麦后其蛋白质和酯酶同工酶分析

对由离子束介导大豆DNA导入豫麦52获得的第四代两个转化株系9004和9002中的可育株籽粒和矮秆不育株杂交籽粒以及受体豫麦52籽粒,进行酯酶和可溶性蛋白的聚丙烯酰胺凝胶电泳分析。结果表明,可育株籽粒可溶性蛋白电泳在谱带上与受体存在差异,矮秆不育株材料的酯酶和可溶性蛋白电泳图谱谱带差异更明显。由此推测,外源大豆DNA导入受体后某些片段有可能插入受体基因组,从而导致基因突变或受体基因表达发生改变。     

玉米酯酶同工酶和过氧化物酶同工酶的分子量研究

本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。     

以靛酚乙酸酯为底物的酯酶同工酶显色方法的研究

建立一种以靛酚乙酸酯为底物的酯酶同工酶的显色新方法。酯酶样品的聚丙烯酰胺凝胶电泳（PAGE）凝胶用磷酸缓冲液漂洗约10min后,浸入含有0.002%靛酚乙酸酯的溶液显色5～10min,可显出清晰的蓝色酯酶带。先将酯酶凝胶板浸于有机磷农药溶液中,然后再用靛酚乙酸酯显色液显色,比较同工酶谱,从同工酶带由深蓝色变为浅蓝色的颜色变化,可以看出对有机磷农药敏感的同工酶所受到的抑制程度。