Cell division and cell enlargement in fruit of Lagenaria leucantha as influenced by pollination and plant growth substances

The effects of NAA (naphthaleneacetic acid), GA₃ (gibberellic acid), CPPU (N-(2-chloro-4-pyridyl)-N'-phenylurea) and pollination on fruit set, cell division and enlargement were studied in Lagenaria leucantha, an important vegetable. NAA and GA₃ were ineffective in inducing parthenocarpy, whereas CPPU induced parthenocarpic fruit significantly larger than fruit that resulted from pollination. Cell division, which occurred during the first 4 days after pollination was not reactivated by NAA or GA₃, but was effectively reactivated by CPPU. The cell number of the total cross-section of CPPU-treated fruit was 117.4% of that of pollinated fruit and 154.4% of that of unpollinated at 12 DAA (days after anthesis) respectively. The CPPU-induced parthenocarpic fruit had the largest cell cross-sectional area followed, successively, by pollinated fruit, NAA-treated fruit, GA₃-treated fruit and unpollinated fruit. These results indicate that CPPU induced parthenocarpic fruit growth by directly reactivating cell division and expansion.     

Correlation of spermine levels with ovary senescence and with fruit set and development inPisum sativum L.

Separation and quantitation of polyamines from unpollinated pea (Pisum sativum L.) ovaries and young fruits induced by application of gibberellic acid to unpollinated ovaries showed, in both cases, a decrease in putrescine and spermidine levels between anthesis and 4 d later. By contrast, spermine levels increased prior to the onset of senescence of the unpollinated ovaries (3 d post anthesis) and decreased during fruit development. Low levels of putrescine, spermidine and spermine were also observed in young fruits obtained by self-pollination and by treatment of unpollinated ovaries with 2,4-dichlorophenoxyacetic acid. In-vitro culture of ovary explants in a medium containing spermine showed that a reduction of the growth of gibberellic acid-treated unpollinated ovaries was associated with a rise in the level of spermine in the fruits. The results obtained indicate that changes in spermine levels are involved in the control of ovary senescence and of fruit set and development.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophen-oxyacetic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography     

Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Changes in the structure of ovary tissues and in the ultrastructure of mesocarp cells during ovary senescence or fruit development induced by plant growth substances in Pisum sativum

Structural changes of tissues in unpollinated ovaries of Pisum sativum L. cv. Alaska after treatment with different plant growth substances (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and 6-benzyladenine) or decapitation of the plant were studied. All the treatments resulted in the prevention of cellular disorganization associated with ovary senescence. They effected the enlargement of mesocarp cells and the differentiation of endocarp cells in very similar patterns, suggesting a similar induction of the structural processes involved in fruit development. Ultrastructural changes in mesocarp cells after treatment with gibberellic acid showed that rapid enlargement of mesocarp cells was sustained mainly by a reorganization of the membrane systems directed to the sysnthesis of primary cell wall. Early changes in the subcellular components in mesocarp cells were observed as the first symptoms in ovary senescence.     

Cloning and Molecular Analysis of cDNA Encoding a Carboxymethylcellulase of the Yeast Cryptococcus flavus

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.     

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.     

Intercellular Localization of Acid Invertase in Tomato Fruit and Molecular Cloning of a cDNA for the Enzyme

The localization of acid invertase (AI, EC 3.2.1.26 [EC] ) in tomatofruits was studied. AI was localized in the intercellular fraction(cell wall fraction). A cDNA encoding a wall-bound form of AIfrom tomato fruits was cloned and its nucleotide sequence wasdetermined. The cloned cDNA was 2363 base pairs long and containedan open reading frame of 1908 base pairs which encoded a polypeptideof 636 amino acids. RNA blot analysis indicated that the mRNAfor the acid invertase was about 2.5 kb in length. The levelsof the mRNA were low at the mature green stage but increasedduring ripening of fruit. (Received July 13, 1992; Accepted December 3, 1992)     

Isolation and characterization of acid invertase cDNA clone in hot pepper (Capsicum annuum L.) fruits

An acid invertase (EC 3.2.1.26.) cDNA clone,CaAIV-18, was isolated from the red pericarp cDNA library of the hot pepper (Capsicum annuum L.) fruit. TheCaAIV-18 clone has 2223 nucleotides and one open reading frame encoding 641 amino acid residues. Analysis of deduced amino acid sequences reveals thatCaAIV-18 has a 24-amino acid transmembrane anchor region in its N-terminal, implying acid invertase in hot pepper may be localized in the membrane and not in the cytosol. This clone showed high homology to tomato acid invertase,Aiv1, in nucleotide and deduced amino acid sequences. In the Southern blot analysis, this clone proved to exist as single or low copy numbers on the genome of hot pepper. The clones had two well-conserved regions which appears in acid invertase of other plant species (eg. tomato,Arabidopsis, etc.) and yeasts. During fruit development,CaAIV-18 was expressed preferentially in the ripe red stage.     

罗汉果色氨酸转氨酶基因SgTAR2的克隆及表达分析

色氨酸转氨酶基因家族,是直接参与植物生长素生物合成途径的关键酶基因。该研究在罗汉果转录组测序的基础上,结合RACE技术克隆罗汉果色氨酸转氨酶基因SgTAR2的全长cDNA序列和DNA序列;并对其进行生物信息学分析和时空表达分析。结果表明:克隆所得SgTAR2的cDNA全长序列2078bp,最长ORF为1332bp,编码443个氨基酸,Gen Bank登录号为KU949381,其编码蛋白具有2个蒜氨酸酶保守结构域和多个5'-PLP结合位点,推测其可能参与催化色氨酸转氨基作用、化学防御作用、生长素生物合成等生物学过程;SgTAR2基因DNA长为4103bp,含有4个内含子和5个外显子,其内含子具有多个高水平转录调控因子和多个与激素、环境等胁迫响应相关的作用元件,暗示SgTAR2基因内含子协同调控罗汉果生长素合成、抗胁迫反应、形态发育等生物学过程。实时荧光定量结果显示,SgTAR2基因在罗汉果各组织器官均有表达,在雌蕊和15d幼果期表达量较高,暗示该基因参与罗汉果果实早期发育。该研究结果表明SgTAR2参与了生长素介导的罗汉果不同生长发育过程,特别对幼果及花的起始发育和器官形态建成等具有重要意义。     

甜瓜乙烯应答因子基因在果实发育成熟过程中的表达特性

根据已报道的甜瓜CMe-ERF1和CMe-ERF2基因cDNA序列设计合成特异性引物,应用RT-PCR技术从甜瓜品种‘河套蜜瓜’成熟果实中克隆得到CMe-ERF1和CMe-ERF2基因cDNA全长编码区序列,分别为498bp和822bp.序列比对分析表明,得到的cDNA序列与已报道的Andes甜瓜相应基因的cDNA序列完全一致.果实不同发育时期实时定量PCR检测结果表明,CMe-ERF1、CMe-ERF2基因表达与甜瓜果实成熟及乙烯生成量显著相关,表明该基因可能对果实成熟起重要作用.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Differential regulation of the tomato ETR gene family throughout plant development

Ethylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ～ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue-specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.     

Induction of fruit set and development in pea ovary explants by gibberellic acid

The response of unpollinated ovary explants ofPisum sativum L. cv. Alaska No. 7 to several plant growth regulators and nutrients has been studied. Explants consisted of a segment of stem and an emasculated flower with or without the adjacent leaf. They were made on the day equivalent to anthesis and were cultured in a liquid medium. Growth regulators were applied either in the solution or directly to the ovaries. Giberellic acid (GA₃) in the presence of sucrose, but not indole-3-acetic acid or N⁶-(Δ²-isopentenyl)-adenine (2iP), induced fruit set and development of parthenocarpic fruits, the final length of these being a function of the intensity of the GA₃ treatment. The capacity of ovaries to respond fully to GA₃ was not lost after incubation of explants in water or 50 mM sucrose for 1 day and was similar in explants made between the day of anthesis and 3 days later. Limited growth was obtained with 100 mM sucrose alone but this effect was counteracted by 2′-isopropyl-4′-(trimethyl ammonium chloride)-5′-methylphenyl piperidine-1-carboxylate (AMO-1618). This inhibitor was ineffective when GA₃ was applied to the ovary. The development of the fruit was proportional to the length of the segment of stem up to 5 cm. The presence of the leaf in the explant enhanced the development of the fruit. These results indicate that a gibberellin is necessary for setting and development of fruits from cultured ovaries and that this effect depends on an appropriate source of nutrients. The course of development of parthenocarpic fruits on explants was similar to that of seeded fruits on the intact plant. The cultured pea ovary systemoffers convenient means to investigate the role of gibberellins and nutrients in fruit set and development.     

Expression of thiol proteases decreases in tomato ovaries after fruit set induced by pollination or gibberellic acid

The modulation of the expression of thiol proteases during both senescence and development was investigated Proteolytic activity and some thiol proteases were analyzed in unpollinated tomato (Lycopersicon esculentum Mill. cv. Rutgers) ovaries during presenescence and during early fruit development induced by treatment with gibberellic acid (GA) or by natural pollination. Proteolytic activity in extracts was tested on azocasein and by observing degradation of the ribulose-1,5-bisphosphate carboxylase large subunit in western blots. There was no correlation between total activity and protein content. Thiol proteases were analyzed by western blot with antibodies raised against papain and a recombinant tomato C14 thiol protease. A 58-kDa polypeptide was recognized by both antibodies and two more polypeptides of 47 and 36 kDa were detected with the second one. All these polypeptide levels increased in untreated unpollinated ovaries at the presenescent stage. Natural pollination or GA treatment of unpollinated ovaries resulted in decreases of these polypeptides at an early developmental stage. The same pattern was observed for the levels of C14 mRNA. Our results suggest that the expression of C14 thiol protease occurs in unpollinated ovaries at the presenescent stage and that it can be suppressed by factors that induce fruit set and development.     

Hormonal Control of Parthenocarpic Ovary Growth by the Apical Shoot in Pea

The role of the apical shoot as a source of inhibitors preventing fruit growth in the absence of a stimulus (e.g. pollination or application of gibberellic acid) has been investigated in pea (Pisum sativum L.). Plant decapitation stimulated parthenocarpic growth, even in derooted plants, and this effect was counteracted by the application of indole acetic acid (IAA) or abscisic acid (ABA) in agar blocks to the severed stump. The treatment of unpollinated ovaries with gibberellic acid blocked the effect of IAA or ABA applied to the stump. [³H]IAA and [³H]ABA applied to the stump were transported basipetally, and [³H]ABA but not [³H]IAA was also detected in unpollinated ovaries. The concentration of ABA in unpollinated ovaries increased significantly in the absence of a promotive stimulus. The application of IAA to the stump enhanced by 2- to 5-fold the concentration of ABA in the inhibited ovary, whereas the inhibition of IAA transport from the apical shoot by triiodobenzoic acid decreased the ovary content of ABA (to approximately one-half). Triiodobenzoic acid alone, however, was unable to stimulate ovary growth. Thus, in addition to removing IAA transport from the apical shoot, the accumulation of a promotive factor is also necessary to induce parthenocarpic growth in decapitated plants.     

Cloning of a vacuolar invertase from Belgian endive leaves (Cichorium intybus)

Although a lot of vacuolar invertase (EC 3.2.1.26) cDNAs are available from a diversity of plant species, up to now no sequence information is available on invertases from any dicot fructan-containing species. Therefore, we describe the cloning of vacuolar acid invertase cDNA from etiolated Belgian endive leaves ( Cichorium intybus L. var. foliosum cv. Flash), formed throughout the forcing process of the witloof chicory roots. Full-length cDNA was obtained by a combination of RT-PCR, PCR and 5'- and 3' RACE RT-PCR, starting with primers based on conserved amino acid sequences. The cloned chicory acid invertase groups together with vacuolar type invertases and fructan biosynthetic enzymes. A putative role for vacuolar type invertases in fructan synthesizing plants is discussed.