A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control

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Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12–24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the -glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.     

Expression profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots: analysis by real-time RT-PCR

BACKGROUND AND AIMS Mineral nutrient deficiencies and salinity constitute major limitations for crop plant growth on agricultural soils. 14-3-3 proteins are phosphoserine-binding proteins that regulate the activities of a wide array of targets via direct protein-protein interactions and may play an important role in responses to mineral nutrients deficiencies and salt stress. In the present study, the expression profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots was investigated in order to analyse the 14-3-3 roles of the proteins in these abiotic stresses. METHODS: Sequence identities and phylogenetic tree creation were performed using DNAMAN version 4.0 (Lynnon Biosoft Company). Real-time RT-PCR was used to examine the expression of each 14-3-3 gene in response to salt stress and potassium and iron deficiencies in young tomato roots. KEY RESULTS: The phylogenetic tree shows that the 14-3-3 gene family falls into two major groups in tomato plants. By using real-time RT-PCR, it was found that (a) under normal growth conditions, there were significant differences in the mRNA levels of 14-3-3 gene family members in young tomato roots and (b) 14-3-3 proteins exhibited diverse patterns of gene expression in response to salt stress and potassium and iron deficiencies in tomato roots. CONCLUSIONS: The results suggest that (a) 14-3-3 proteins may be involved in the salt stress and potassium and iron deficiency signalling pathways in young tomato roots, (b) the expression pattern of 14-3-3 gene family members in tomato roots is not strictly related to the position of the corresponding proteins within a phylogenetic tree, (c) gene-specific expression patterns indicate that isoform-specificity may exist in the 14-3-3 gene family of tomato roots, and (d) 14-3-3 proteins (TFT7) might mediate cross-talk between the salt stress and potassium and iron-deficiency signalling pathways in tomato roots.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

缺铁胁迫下4种果树砧木中三价铁螯合物还原酶基因的表达

用活力染色法观测 4种果树砧木中FCR基因表达的结果表明 ,小金海棠 (M .xiaojinensis)和香橙 (C .junos)在缺铁胁迫下根吸收区FCR活性明显增强 ,增强幅度显著强于丽江山荆子 (M .rockii)和枳 (P .trifoliata)。用拟南芥的FCR基因 (FRO2 )做探针 ,进行组织印迹的RNANorthern杂交。结果表明 ,在缺铁胁迫下 ,在小金海棠和香橙中均有与FRO2类似基因的强烈表达 ,而在同样胁迫条件下的枳和丽江山荆子中表达却很微弱。这种分子水平上的反应与通常所熟知的生理反应是一致的 ,说明小金海棠和香橙忍耐缺铁环境的生理机制和分子基础类似 ,FCR在它们忍耐缺铁的生化反应中起着非常重要的作用 ,FCR基因在 4种果树砧木中的表达水平高低与它们忍耐缺铁的能力呈现正相关。并且 ,在小金海棠和香橙的根、茎、叶等营养器官的特定组织细胞中 ,均能检出高水平的FCR基因转录产物 ,表明耐缺铁能力强的小金海棠和香橙体内存在高效的铁运输和利用机制     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

AIMS: Isolation of the genes relative to PCB biodegradation and identification of the bph gene function in Rhodococcus sp. R04. METHODS AND RESULTS: A 8.7-kb fragment carrying the biphenyl catabolic genes bphABCD was isolated from the gene library in Rhodococcus sp. R04. Based on the deduced amino acid sequence homology, seven bph genes, bphA1A2A3A4, bphB, bphC and bphD, were thought to be responsible for the initial four steps of biphenyl degradation. In Escherichia coli, BphA exhibited poor activity for biphenyl transformation, and BphB, BphC and BphD were found to be catalytically active towards 2,3-dihydro-2,3-dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, respectively (activities of 50, 8.1 and 2.4 micromol l(-1) min(-1) mg(-1)). SDS-PAGE analysis indicated that the sizes of bphA1A2A3A4, bphB, bphC and bphD gene products were 49, 19, 14, 47, 32, 30 and 31 kDa, respectively. After disruption of bph genes, the bphA1 mutants lost the ability to grow on biphenyl, the bphB and bphD mutants were able to transform a little of biphenyl, but hardly grew on biphenyl. CONCLUSION: The cloned bph genes indeed play an important role in the biphenyl catabolism in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This bph gene organization in Rhodococcus sp. R04 differs from that of other biphenyl degraders reported previously, indicating it is a novel type of bph gene cluster. Analysis of the phylogenetic tree suggested that BphA1 and BphA2 in Rhodococcus sp. R04 had a different evolutionary relationship with those in the other PCB degraders.     

Salt and paraquat stress tolerance results from co-expression of the Suaeda salsa glutathione S-transferase and catalase in transgenic rice

GST (Glutathione S-transferase, EC 2.5.1.18) and CAT (Catalase, EC 1.11.1.6) play important roles in oxidative stress resistance. In this study, we transferred both GST and CAT1 of Suaeda salsa into rice (Oryza sativa cv. Zhonghua No.11) by Agrobacterium tumefaciens-mediated transformation under the control of cauliflower mosaic virus (CaMV) 35S promoter, and investigated whether co-expressing the GST and CAT1 in transgenic rice could reduce oxidative damage. Salt and paraquat stresses were applied. The data showed that co-expression of the GST and CAT1 resulted in greater increase of CAT and SOD (Superoxide Dismutase, EC 1.15.1.1) activity in the transgenics compared to non-transgenics following both stress imposition. Whereas the significant increase of GST activity in transgenics only occurred in paraquat stressed plants. While the generation of H₂O₂, Malon dialdehyde and plasma membrane relative electrolyte leakage decreased in the transgenics than in non-transgenics under the same conditions. Moreover, the transgenic rice seedlings showed markedly enhanced tolerance to salt stress compared with non-transgenics upon 200 mM NaCl treatment in greenhouse. The enhancement of the active oxygen-scavenging system that led to increased oxidative stress protection in GST + CAT1-transgenic rice plants could result not only from increased GST and CAT activity but also from the combined increase in SOD activity.     

油菜APETALA3基因5′调控序列的克隆及分析

利用单引物PCR方法获得了甘蓝型油菜APETALA3重复基因拷贝BnAP3-3和BnAP3-4的5' 调控序列.分析结果表明,两段调控区序列一致性为80.43%,大多数的保守功能元件如TATA盒和CAAT盒没有明显差异,都含有一些应对环境胁迫及诱导的元件,如MYB1AT、GATABOX和DOFCOREZM等.少数顺式作用元件存在较大差异,其中CGACGOSAMY3、CArG box、CARGCW8GAT及CAREOSREP1为BnAP3-3的5'调控序列所特有,而BnAP3-4的5'调控序列则含有GT1CONSENSUS.半定量RT-PCR显示,BnAP3-3表达量远高于BnAP3-4,可能与二者调控区某些功能元件序列差异有关.     

Expression analysis of the two ferrochelatase genes in Arabidopsis in different tissues and under stress conditions reveals their different roles in haem biosynthesis

The Arabidopsis thaliana genome has two genes (AtFC-I and AtFC-II), encoding ferrochelatase, the terminal enzyme of haem biosynthesis. The roles of the two enzymes in the synthesis of haem for different haemoproteins was investigated using reporter gene analysis. A 1.41 kb fragment from the 5' upstream region of the AtFC-II gene was fused to the luciferase gene, and then introduced into tobacco plants, followed by luciferase activity measurements. AtFC-II-LUCwas expressed in all aerial parts of the plant, and was highest in flowers, but it was not expressed in roots. It was unaffected by viral infection, and considerably reduced by wounding or oxidative stress. Similarly, a 1.76 kb region of the AtFC-I promoter was fused to the uidAgene encoding -glucuronidase. AtFC-I-GUS was expressed in all tissues of the plant, but was higher in roots and flowers than in leaves or stems. It was induced by sucrose, wounding and oxidative stress and, most markedly, by plants undergoing the hypersensitive response to TMV infection. Levels of endogenous ferrochelatase activity were increased in pea chloroplasts isolated from wounded leaves, indicating that the induction in promoter activity is likely to result in increased haem biosynthetic potential. Salicylic acid, but not methyl-jasmonate was able to replace the stress treatment in induction of AtFC-I expression, suggesting that the requirement for haem synthesis is part of the defence response. The implications of the results for the different roles of the two ferrochelatases in haem biosynthesis are discussed.     

Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron (Crocus sativus L.)

Physiology and Molecular Biology of Plants - Crocus sativus, a monocot triploid species belonging to the Iridaceae family, is cultivated for its red stigmatic lobes of the carpel that constitute...     

Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii

Ferritin is a key player in the iron homeostasis due to its ability to store large quantities of iron. Chlamydomonas reinhardtii contains two nuclear genes for ferritin ( ferr1 and ferr2 ) that are induced when Chlamydomonas cells are shifted to iron-deficient conditions. In response to the reduced iron availability, degradation of photosystem I (PSI) and remodeling of its light-harvesting complex occur. This active PSI degradation slows down under photo-autotrophic conditions where photosynthesis is indispensable. We observed a strong induction of ferritin correlated with the degree of PSI degradation during iron deficiency. The PSI level can be restored to normal within 24 h after iron repletion at the expense of the accumulated ferritin, indicating that the ferritin-stored iron allows fast adjustment of the photosynthetic apparatus with respect to iron availability. RNAi strains that are significantly reduced in the amount of ferritin show a striking delay in the degradation of PSI under iron deficiency. Furthermore, these strains are more susceptible to photo-oxidative stress under high-light conditions. We conclude that (i) ferritin is used to buffer the iron released by degradation of the photosynthetic complexes, (ii) the physiological status of the cell determines the strategy used to overcome the impact of iron deficiency, (iii) the availability of ferritin is important for rapid degradation of PSI under iron deficiency, and (iv) ferritin plays a protective role under photo-oxidative stress conditions.     

Promoter activation of pepper class II basic chitinase gene, CAChi2, and enhanced bacterial disease resistance and osmotic stress tolerance in the CAChi2-overexpressing Arabidopsis

The activation of the CAChi2 promoter as the result of bacterial infection and osmotic stresses was examined using the Agrobacterium-mediated transient expression assay. Several stress-related cis-acting elements were revealed within the upstream genomic sequence of the CAChi2 gene. In tobacco leaf tissues transiently transformed with the CAChi2 promoter-β-glucuronidase (GUS) gene, the CAChi2 promoter was up-regulated by Pseudomonas syringae pv. tabaci infection. The CAChi2-GUS activation was closely related to osmotic stresses, including treatment with mannitol and NaCl. The −378 CAChi2 promoter was sufficient for the CAChi2 gene induction by salicylic acid treatment. CAChi2 overexpression in the transgenic Arabidopsis plants enhanced bacterial disease resistance against Pseudomonas syringae pv. tomato infection. CAChi2-overexpressing Arabidopsis plants also exhibited increased tolerance to NaCl-induced osmotic stresses during seed germination and seedling growth. CAChi2 overexpression induced the expression of the NaCl stress-responsive gene RD29A in the absence of NaCl stress. The CAChi2-overexpressing transgenic plants exhibited increased sensitivity to abscisic acid during seed germination. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number AY775335.     

Isolation of Xenopus HGF gene promoter and its functional analysis in embryos and animal caps

Previously, we isolated Xenopus HGF (hepatocyte growth factor) cDNA and showed in Xenopus embryos that expression of this gene starts at the late gastrula stage mainly in the ventral mesoderm, and furthermore that the expression is induced in animal cap by activin A and bFGF (basic fibroblast growth factor). Here we have cloned the Xenopus HGF gene, covering a 14 kb 5-upstream region and a 0.2 kb 5-coding region. Within about 0.5 kb of the 5-flanking region, the Xenopus HGF gene contained a TATA-like element AATGAAA, one putative NF-1 binding site, two NF-IL-6 binding motif sequences, one putative TGF--dependent inhibitory element (TIE) and one AP-1 binding site. A recombinant circular plasmid consisting of a 1.7 kb HGF promoter region and the bacterial chloramphenicol acetyltransferase (CAT) gene was first expressed at the late gastrula stage in the ventral mesoderm, as was the endogenous HGF gene. The expression of the fusion gene was induced in animal cap cells by activin A and bFGF although induction by the latter was not so strong. Using a series of 5-deletion constructs introduced into animal caps, silencer elements, which seem to be essential for the gene's regionally correct expression, and the element responsible for induction by activin were found. The results show that the HGF gene promoter isolated here contains elements which may endow the gene with the regulative function for its temporally and spatially regulated expression, although the element necessary for induction by bFGF seems to be missing.     

Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.     

Molecular cloning,characterization and expression analysis of BcHHP3 under abiotic stress in Pak-choi (Brassica rapa ssp. Chinensis)

In this study, BcHHP3 was isolated from Pak-choi; it has an open-reading frame (ORF) of 1044 base pairs, encoding 347 amino acids, a molecular weight of 39.35?kDa, isoelectric point (pI) of 9.08, an instability index of 48.35, and grand average of hydropathicity of 0.382. Multi-alignment and phylogenetic analysis showed that BcHHP3 bears a high similarity to AtHHP2. As predicted by SOMPA and SWISS-MODEL databases, the structure of the BcHHP3 protein is relatively stable and highly conservative. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that BcHHP3 was induced to co-express under cold and abscisic acid (ABA) stresses. The BcHHP3-GFP fusion protein was localized on the cell membrane and nuclear membrane. This work might be useful for future analysis of other HHP-like genes in Pak-choi.