Detection of 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein Cm-HMGR during fruit development in melon ( Cucumis melo L.)

The fruit size of melon (Cucumis melo L. reticulatus) is determined by the amount of cell proliferation in the pericarp during early fruit development. During this stage, expression and activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene is required for fruit growth. In this study, we performed a detailed analysis of the correlation between the expression of melon HMGR (Cm-HMGR) protein and cell division in the pericarp. Flow cytometric analysis revealed that the length of the cell division stage was correlated with the fruit size. Western gel blotting and tissue printing illustrated the temporal and spatial accumulation pattern of Cm-HMGR protein during fruit development. The accumulation of Cm-HMGR transiently increased at the beginning of the cell division stage in the pericarp, where active cell division occurred. The amount of Cm-HMGR was correlated with the length of the cell division period. These results strongly suggest that the expression of Cm-HMGR is involved in the determination of melon fruit size by regulating cell division during early fruit development.     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.     

植物3-羟基-3-甲基戊二酰辅酶A还原酶基因研究进展

细胞分裂素、赤霉素、脱落酸、叶绿素、萜类等类异戊二烯物质,是植物中广泛存在的一类代谢产物,在植物生长发育过程中起着非常重要的作用。一些萜类化合物作为药物的合成前体或有效的药用成分在工农业及医药生产上具有重要的经济价值。类异戊二烯物质主要通过甲羟戊酸代谢途径中的一系列酶催化合成,其中,3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是该代谢途径中的第一个关键限速酶,能够将3-羟基-3-甲基戊二酰辅酶A转化成中间代谢产物甲羟戊酸。对植物HMGR基因的克隆、酶结构和功能分析、基因组织表达及调控等方面进行了综述,旨在为其在重要农作物的遗传改良、代谢产物工程植物创制以及植物亲缘关系分析中的应用等研究提供理论依据。     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

细纹豆芫菁3-羟甲基戊二酰辅酶A-还原酶基因全长cDNA克隆及生物信息学分析

3-羟甲基戊二酰辅酶A-还原酶（3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR）是甲羟戊酸途径的关键酶。获得芫菁体内HMGR基因信息是确定甲羟戊酸途径与斑蝥素合成相关性的基础。本研究利用RACE技术从细纹豆芫菁Epicauta mannerheimi (Maklin)体内克隆获得HMGR基因全长cDNA序列, 命名为EmHMGR（GenBank登录号为JQ690539）。该基因全长3 118 bp, 其中5′端非翻译区178 bp, 3′端非翻译区414 bp, 开放阅读框2 526 bp, 编码842个氨基酸。推测的蛋白质分子量为92.8 kDa, 理论等电点为6.0, 预测分子式为C₄₁₃₅H₆₆₀₄N1₀₉₈O₁₂₁₆S₅₀, 不稳定系数为43.37, 总亲水性系数为0.091, 为疏水性不稳定蛋白。序列分析发现该基因编码的蛋白与已报道的其他昆虫HMGR的氨基酸序列一致性达50%以上, 而且包含HMGR_Class I保守功能域、 固醇敏感多肽区及HMGR蛋白的其他保守功能位点。系统进化分析发现该基因与叶甲科昆虫HMGR基因的关系最近。本研究首次从芫菁科昆虫体内克隆获得甲羟戊酸途径的关键酶EmHMGR基因, 为后期芫菁体内斑蝥素生物合成途径的研究奠定了基础。     

SfHMGR在白背飞虱生殖调控中的功能分析

[目的]为探究保幼激素(juvenile hormone,JH)生物合成关键基因3-羟基-3-甲基戊二酰辅酶A还原酶基因(HMGR)在白背飞虱Sogatella furcifera生殖中的作用.[方法]基于已公布的白背飞虱基因组和转录组数据,结合RT-PCR技术获得SfHMGR全长cDNA序列;采用RT-qPCR技术分...     

Fruit size: Towards an understanding of the metabolic control of fruit growth using avocado as a model system

Final fruit size is the consequence of complex metabolic events that occur between fruit set and maturation. Disruption of these biochemical and molecular processes at any stage during fruit growth will impact on final fruit size. Because fruit size is a function of cell number rather than cell size, factors affecting cell division cycle activity assume importance. In this paper, we focus attention on the metabolic control of fruit growth using avocado as a model system. Three areas of current interest are highlighted, viz. the contribution by isoprenoid metabolism in the control of cell proliferation, the role played by carbohydrate content and composition in signalling changes in metabolite status and gene expression and maintenance of plant hormone homeostasis. Central to the process of fruit growth and control of final fruit size by cell division is 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and activity of the sucrose non-fermenting 1-related protein kinase (SnRK1) complex. It is argued that sugar content and composition of sink cells impact on SnRK1 (and hexokinase) to modulate expression of sugar-metabolizing enzymes, HMGR and molybdenum cofactor (MoCo)-containing enzymes. These changes, in turn, impact on hormone metabolism by affecting allocation of the purine-derived MoCo to aldehyde oxidase and thus the endogenous concentration of indole-3-acetic acid, abscisic acid and cytokinin (CK) to alter plant hormone homeostasis. These aspects are integrated into a model to explain the metabolic control of avocado fruit growth and final fruit size.     

Differential expression of litchi XET genes in relation to fruit growth

Xyloglucan endotransglycosylase (XET) catalyses the transglycosylation of xyloglucan, the major hemicellulose polymer, which has been thought to mediate the cross-linking of cellulose microfibrils in cellular walls and proposed to be involved in the control of cell wall relaxation. To understand the relationship between litchi fruit cracking and gene expression patterns, three XET genes from litchi fruit were identified and then examined for their expression profiles in pericarp and aril tissues at different development stages, using a cracking-resistant cultivar, 'Huaizhi', and a cracking-susceptible cultivar, 'Nuomici'. Three full-length cDNAs of 1267, 1095 and 1156 bp encoding XETs, named LcXET1, LcXET2 and LcXET3, respectively, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Northern blotting analysis showed that LcXET1 mRNA accumulation occurred much earlier in aril tissues at 59 days after anthesis (DAA) than in pericarp tissues at 73 DAA in 'Nuomici'. However, it appeared at almost the same time (66 DAA) in pericarp and aril tissues in 'Huaizhi', which suggested that differential accumulation of LcXET1 in pericarp and aril tissues in 'Nuomici' and 'Huaizhi' was closely associated with fruit cracking. LcXET2 mRNA accumulation could be detected in pericarp and aril tissues throughout fruit development but exhibited a differential accumulation pattern between pericarp and aril tissues. In the aril of 'Nuomici', intensive signal bands were detectable at 59-73 DAA in rapidly expanding fruits of 'Nuomici' but only weak bands could be found in the pericarp tissues. In contrast, moderate signal bands were detectable both in pericarp and aril tissues of 'Huaizhi' fruits. Furthermore, LcXET3 showed constitutive expression in both pericarp and aril tissues of developing 'Nuomici' and 'Huaizhi' litchi fruit. In addition, differential expression patterns of three XETs genes were observed in different tissues of litchi, with only LcXET1 being fruit-specific. To further address the role of LcXET in fruit cracking, alpha-naphthalene acetic acid (NAA) was used to treat 'Nuomoci' to reduce fruit cracking. Enhanced LcXET1 mRNA accumulation appeared in pericarp while LcXET2 and LcXET3 mRNA accumulation enhanced in aril tissues in the NAA-treated fruits. Thus, LcXET1 is more likely to play a role in reducing litchi fruit cracking than LcXET2 and LcXET3.     

A multilevel analysis of fruit growth of two tomato cultivars in response to fruit temperature

Fruit phenotype is a resultant of inherent genetic potential in interaction with impact of environment experienced during crop and fruit growth. The aim of this study was to analyze the genetic and physiological basis for the difference in fruit size between a small (‘Brioso’) and intermediate (‘Cappricia’) sized tomato cultivar exposed to different fruit temperatures. It was hypothesized that fruit heating enhances expression of cell cycle and expansion genes, rates of carbon import, cell division and expansion, and shortens growth duration, whereas increase in cell number intensifies competition for assimilates among cells. Unlike previous studies in which whole‐plant and fruit responses cannot be separated, we investigated the temperature response by varying fruit temperature using climate‐controlled cuvettes, while keeping plant temperature the same. Fruit phenotype was assessed at different levels of aggregation (whole fruit, cell and gene) between anthesis and breaker stage. We showed that: (1) final fruit fresh weight was larger in ‘Cappricia’ owing to more and larger pericarp cells, (2) heated fruits were smaller because their mesocarp cells were smaller than those of control fruits and (3) no significant differences in pericarp carbohydrate concentration were detected between heated and control fruits nor between cultivars at breaker stage. At the gene level, expression of cell division promoters (CDKB2, CycA1 and E2Fe‐like) was higher while that of the inhibitory fw2.2 was lower in ‘Cappricia’. Fruit heating increased expression of fw2.2 and three cell division promoters (CDKB1, CDKB2 and CycA1). Expression of cell expansion genes did not corroborate cell size observations.     

Cloning and Characterization of a Root-specific Expressing Gene Encoding 3-hydroxy-3-methylglutaryl Coenzyme a Reductase from Ginkgo biloba

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34) catalyzes the first committed step in mevalonic acid (MVA) pathway for biosynthesis of isoprenoids. The full-length cDNA encoding HMGR was isolated from Ginkgo biloba for the first time (designated as GbHMGR, GenBank accession number AY741133), which contained a 1713 bp ORF encoding 571 amino acids. The GbHMGR genomic DNA sequence was also obtained, revealing GbHMGR had four exons and three introns. The deduced GbHMGR protein showed high identity to other plant HMGRs and contained two trans-membrane domains and a catalytic domain. The three dimensional model of GbHMGR represented a typical spatial structure of HMGRs. The Southern blot and RT-PCR assay results indicated that GbHMGR belonged to a small gene family, and expressed in a tissue-specific manner with a low level expression being only found in root. The potential significance of GbHMGR gene was also discussed.     

Pollination: A key event controlling the expression of genes related to phytohormone biosynthesis during grapevine berry formation

Berry formation is the process of ovary conversion into a functional fruit, and is characterized by abrupt changes in the content of several phytohormones, associated with pollination and fertilization. Much effort has been made in order to improve our understanding of berry development, particularly from veraison to post-harvest time. However, the period of berry formation has been poorly investigated, despite its importance. Phytohormones are involved in the control of fruit formation; hence it is important to understand the regulation of their content at this stage. Grapevine is an excellent fleshy-fruit plant model since its fruits have particularities that differentiate them from those of commonly studied organisms. For instance, berries are prepared to cope with stress by producing several antioxidants and they are non-climacteric fruits. Also its genome is fully sequenced, which allows to identify genes involved in developmental processes. In grapevine, no link has been established between pollination and phytohormone biosynthesis, until recently. Here we highlight relevant findings regarding pollination effect on gene expression related to phytohormone biosynthesis, and present results showing how quickly this effect is achieved.     

Cell number, cell size and hormone levels in semi-isogenic mutants of Lycopersicon pimpinellifolium differing in fruit size

Tomato fruit growth parameters, cell number and cell size, and hormone levels [IAA, abscisic acid (ABA), zeatin (Z)/zeatin riboside (ZR), isopentenyladenosine (i-Ado)/isopentenlyadenine (i-Ade)], in the wild-type ( Lycopersicon pimpinellifolium Mill.) and a semi-isogenic mutant (mutant III) differing in fruit size were investigated during fruit development. An image-processing system was used for the determination of cell number and single cell size per fruit and hormone levels were measured by radioimmuno-assay (RIA). The bigger fruits of mutant III showed higher cell numbers throughout fruit development and cells enlarged faster than in wild-type fruits. During the first 10 days of fruit growth, the main cell division period after fertilization, high concentrations of cytokinins were found, these being correlated with high cell division activity. There were only slight differences in IAA and ABA levels in the different sized fruits. The results emphasized the importance of the cell number per fruit at anthesis as a determining factor of final fruit size in tomatoes. A possible relationship between cytokinins and subsequent fruit development is discussed.     

Elucidating the functional role of endoreduplication in tomato fruit development

Background

Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination.

Scope

Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described.

Conclusions

The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.