High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions

The 1.4 kb 5 polygalacturonase (PG) gene-flanking region has previously been demonstrated to direct ripening-specific chloramphenicol acetyl transferase (CAT) expression in transgenic tomato plants. The steady state level of CAT mRNA in these plants was estimated to be less than 1% of the endogenous PG mRNA. Further constructs containing larger PG gene-flanking regions were generated and tested for their ability to direct higher levels of reporter gene expression. A 4.8 kb 5-flanking region greatly increased levels of ripening-specific reporter gene activity, while a 1.8 kb 3 region was only shown to have a positive regulatory role in the presence of the extended 5 region. Transgenic plants containing the CAT gene flanked by both of these regions showed the same temporal pattern of accumulation of CAT and PG mRNA, and steady-state levels of the transgene mRNA were equivalent to 60% of the endogenous PG mRNA on a per gene basis. The proximal 150 bp of the PG promoter gave no detectable CAT activity. However, the distal 3.4 kb of the 4.8 kb 5 PG promoter was shown to confer high levels of ripening-specific gene expression when placed in either orientation upstream of the 150 bp minimal promoter. The DNA sequence of the 3.4 kb region revealed a 400 bp imperfect reverse repeat, and sequences which showed similarity to functionally significant sequences from the ripening-related, ethylene-regulated tomato E8 and E4 gene promoters. The possible roles of the flanking regions in regulating PG gene expression are discussed.     

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential for chloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposed to participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDG occurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substitute DGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthase genes ( MGD1 , MGD2 and MGD3 ), the type-B MGD2 and MGD3 are upregulated in parallel with DGDG synthase genes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertional mutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drastic reduction in DGDG accumulation, particularly in the root, indicating that MGD3 is the main isoform responsible for DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-induced accumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential for membrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosynthetic performance were also observed in these mutants under Pi-starved conditions. These results demonstrate that Pi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. The widespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodeling mediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.     

cDNA structure and regulatory properties of a family of starvation-induced ribonucleases from tomato

In previous work we have determined the primary structure of two of the five ribonucleases which are induced by phosphate starvation in cultured tomato cells. Here, we present the isolation and characterization of the cDNAs for the extracellular ribonuclease LE and the intracellular, but extravacuolar ribonuclease LX. Structural analysis of these cDNAs together with partial protein-sequencing of vacuolar ribonucleases LV1, LV2 and LV3 revealed a family of very similar ribonucleases. From these data we assume identity between ribonucleases LE and LV3 for which the targeting mechanism has to be shown. Furthermore, RNase LV1 and RNase LV2 might be posttranslational processing products of RNase LX which travel to the vacuoles after splitting off the putative ER retention signal present at RNase LX. Additionally, we show by northern blot analysis that phosphate starvation in plant cells leads to an increase in the steady-state level of this type of enzymes revealing close similarities of the plant response to a limited supply of inorganic phosphate with the PHO regulation in bacteria and fungi.     

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings [12]. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the -glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis

Phosphate (P(i)) is a major limiting factor for plant growth. Plants respond to limiting P(i) supplies by inducing a suite of adaptive responses comprising altered growth behaviour, enhanced P(i) acquisition and reduced P(i) demand that together define a distinct physiological state. In P(i)-starved plants, continued root growth is required for P(i) acquisition from new sources, yet meristem activity consumes P(i). Therefore, we analysed the relationship between organ growth, phosphate starvation-responsive (PSR) gene expression and P(i) content in Arabidopsis thaliana under growth-promoting or inhibitory conditions. Induction of PSR gene expression after transfer of plants to P(i)-depleted conditions quantitatively reflects prior levels of P(i) acquisition, and hence is sensitive to the balance of P(i) supply and demand. When plants are P(i)-starved, enhanced root or shoot growth exacerbates, whereas growth inhibition suppresses, P(i) starvation responses, suggesting that the magnitude of organ growth activity specifies the level of P(i) demand. Inhibition of cell-cycle activity, but not of cell expansion or cell growth, reduces P(i) starvation-responsive gene expression. Thus, the level of cell-cycle activity specifies the magnitude of P(i) demand in P(i)-starved plants. We propose that cell-cycle activity is the ultimate arbiter for P(i) demand in growing organs, and that other factors that influence levels of PSR gene expression do so by affecting growth through modulation of meristem activity.     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。     

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.     

A functional map of the fruit-specific promoter of the tomato 2A11 gene

The 5 region of the fruit-specific tomato gene, 2A11, contains both positive and negative regulatory elements. We divided the 5 promoter region of the 2A11 gene into small fragments, ranging in size from 211 to 634 bp and used these short DNA fragments in in vitro protein-binding studies. These studies revealed the presence of at least four fruit-specific and one leaf- and fruit-active protein-binding domains. These promoter fragments, as well as other overlapping fragments, were tested for their ability to enhance expression from a truncated heterologous promoter in transgenic plants. This analysis showed the presence of four fruit-specific and three general or leaf-active positive regulatory elements. Comparison of the results obtained with these two approaches allowed us to draw a functional map of the 2A11 promoter.     

Domestication rewired gene expression and nucleotide diversity patterns in tomato

Plant domestication has led to considerable phenotypic modifications from wild species to modern varieties. However, although changes in key traits have been well documented, less is known about the underlying molecular mechanisms, such as the reduction of molecular diversity or global gene co‐expression patterns. In this study, we used a combination of gene expression and population genetics in wild and crop tomato to decipher the footprints of domestication. We found a set of 1729 differentially expressed genes (DEG) between the two genetic groups, belonging to 17 clusters of co‐expressed DEG, suggesting that domestication affected not only individual genes but also regulatory networks. Five co‐expression clusters were enriched in functional terms involving carbohydrate metabolism or epigenetic regulation of gene expression. We detected differences in nucleotide diversity between the crop and wild groups specific to DEG. Our study provides an extensive profiling of the rewiring of gene co‐expression induced by the domestication syndrome in one of the main crop species.     

磷饥饿对番茄幼苗H+分泌及Pi吸收的影响

磷饥饿条件下番茄幼苗的H⁺分泌速率明显提高。质膜质子泵专一性抑制剂钒酸盐能显著抑制番茄幼苗的H⁺分泌,也能显著抑制其Pi吸收。此结果表明,磷饥饿时番茄幼苗Pi吸收速率的变化与H⁺分泌速率的变化之间可能具有一定的相关性,并进一步暗示质膜H⁺-ATPase可能参与其中。本文结果还表明,Pi/H⁺的准量关系约为1:1。     

Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27°C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32°C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20°C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34°C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20°C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.     

Induction of AGAMOUS gene expression plays a key role in ripening of tomato sepals in vitro

In vitro culture of VFNT Cherry tomato sepals (calyx) at 16–21 °C results in developmental changes that are similar to those that occur in fruit tissue [10]. Sepals become swollen, red, and succulent, produce ethylene, and have increased levels of polygalacturonase RNA. They also produce many flavor volatiles characteristic of ripe tomato fruit and undergo similar changes in sugar content [11]. We examined the expression of the tomato AGAMOUS gene, TAG1, in ripening, in vitro sepal cultures and other tissues from the plant and found that TAG1 RNA accumulates to higher levels than expected from data from other plants. Contrary to reports on the absence of AGAMOUS in sepals, TAG1 RNA levels in green sepals from greenhouse-grown plants is detectable, its concentration increasing with in vitro ripening to levels that were even higher than in red, ripe fruit. Sepals of fruit on transgenic tomato plants that expressed TAG1 ectopically were induced by low temperature to ripen in vivo, producing lycopene and undergoing cell wall softening as is characteristic of pericarpic tissue. We therefore propose that the induction of elevated TAG1 gene expression plays a key role in developmental changes that result in sepal ripening.