Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Level of expression of the tomato rbcS-3A gene is modulated by a far upstream promoter element in a developmentally regulated manner.

By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter sequence from the tomato rbcS-3A gene confers light-inducible and organ-specific expression upon fusion to the bacterial chloramphenicol acetyltransferase gene. A biphasic expression profile was obtained by 5' deletion analysis of this promoter, indicating the presence of both positive and negative regulatory elements. A severe reduction in the level of expression was observed when the 5'-terminal 90 base pairs were deleted from the 1.10-kilobase promoter. DNA sequence elements responsible for light inducibility and organ specificity of the gene reside within the -374 base pairs of the proximal part of the promoter and the sequences spanning from -374 to -205 are essential for promoter function. The DNA sequences upstream from -374 modulate the level of expression in leaf tissue; this modulation is under developmental control.     

A gene duplication/loss event in the ribulose-1,5-bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of Arabidopsis thaliana

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.     

Binding sites for two novel phosphoproteins, 3AF5 and 3AF3, are required for rbcS-3A expression.

Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding sites for the nuclear factor GT-1 within the -166 deleted promoter of the ribulose-1,5-bisphosphate carboxylase-3A (rbcS-3A) gene, suggested that GT-1 might act in concert with an additional protein to confer light-responsive rbcS-3A expression. In this work, S1 analysis of RNA isolated from transgenic tobacco plants carrying mutant rbcS-3A constructs led to the identification of two short sequences located at the 5' and 3' ends of box III that are required for expression. These two sequences serve as binding sites for two novel proteins, 3AF5 and 3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and 3AF3 showed that complexes with faster mobilities were formed using nuclear extracts prepared from dark-adapted plants compared with those from light-grown tobacco plants. Phosphatase treatment of extracts from light-grown plants resulted in the formation of complexes with faster mobility. Although the binding of 3AF3 to its target site is dependent upon phosphorylation, the binding of 3AF5 does not appear to be affected by its phosphorylation state. These results suggest that the phosphorylated forms of both 3AF5 and 3AF3 are required for -166 rbcS-3A expression but that the mechanisms differ by which phosphorylation regulates the activities of 3AF5 and 3AF3.     

The pea rbcS-3A enhancer-like element directs cell-specific expression in transgenic tobacco

Summary The pea rbcS-3A gene codes for the small subunit of ribulose-1,5-bisphosphate carboxylase and its expression is regulated by light, tissue type and stage of development. Analysis in transgenic tobacco plants has shown that the upstream region contains an enhancer-like element which can confer light-regulated and organ-specific expression upon a reporter gene (Fluhr et al. 1986a). Here we address the question of whether the enhancer specifies not only organ specificity, but also expression in the correct cell types. In situ immunofluorescence and microdissection were used on transgenic tobacco plants containing the rbcS-3A enhancer fused to a reporter gene consisting of the cauliflower mosaic virus 35S promoter and the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT levels are high in leaf mesophyll cells, but in the epidermis expression is restricted to guard cells. In the midrib, of the leaf and in the stem, there is considerable signal in the chlorenchyma and in the phloem region. This pattern of expression closely correlates with the distribution of the endogenous RbcS polypeptides and with the presence of chlorophyll. Our results demonstrate that the rbcS-3A enhancer-like element possesses all the necessary DNA sequences for expression in the correct cell types.     

豌豆过氧化氢酶在烟草叶绿体中的过量表达提高了植物的抗逆性

通过构建融合番茄RuBP羧化酶小亚基转运肽基因(rbcS-3)和CAT基因编码阅读框(ORF)的双元表达载体,采用农杆菌介导的叶圆盘转化法将融合基因转入烟草,使其能够定向导入叶绿体中发挥作用。在含有50mg/L潮霉素的培养基上筛选获得转CAT烟草30多个株系,并对其进行了分子生物学的验证和生理指标的检测。对获得的抗性植株用PCR、RT-PCR、植株总蛋白Western blot和叶绿体蛋白Western blot分析表明,目的基因已经整合到烟草基因组中,并能正常表达,且在叶绿体rbcS-3转运肽的作用下能定向进入叶绿体中。对转基因植株生理指标的检测发现,在20% PEG₆₀₀₀模拟干旱条件下,野生型烟草的相对电导率提高幅度为43.4%,而转CAT植株的相对电导率仅提高8.8%,表明在干旱胁迫下转CAT烟草的质膜透性小于野生型烟草;经20% PEG₆₀₀₀处理后,野生型和转CAT基因烟草的叶绿素含量都下降,下降幅度分别为68.0%和20.4%;另外,经20% PEG₆₀₀₀处理的野生型烟草叶片的Fv/Fm下降幅度为5.3%,而转CAT基因烟草叶片的Fv/Fm下降幅度0.9%,这些结果表明,在叶绿体中过量表达CAT对干旱胁迫下的细胞质膜、叶绿素和PSⅡ具有一定的保护作用。此外,经150 μmol/L百草枯处理后发现,处理3h后,野生型烟草和转CAT烟草的相对电导率分别比对照提高67.9%和13.5%,而野生型和转CAT烟草的Fv/Fm都下降,降幅分别为23.7%和3.9%,这表明在百草枯氧化胁迫下转CAT烟草的质膜和PSⅡ的损伤程度都小于野生型烟草。总之,豌豆CAT基因在烟草叶绿体中过量表达,提高了转基因烟草的抗旱性和抗氧化性。     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

Induction and repression of the Drosophila Sgs-3 glue gene are mediated by distinct sequences in the proximal promoter.

The normal developmental expression of the Drosophila salivary gland secretion protein gene Sgs-3 requires the interaction of a distal and proximal regulatory element. A deletion/replacement analysis of the proximal promoter in stably transformed lines shows that induction of an Sgs-3/Adh fusion gene is normal if sequences from +10 to -50 are replaced by those of the hsp70 gene. Sequences between -98 and -50 are necessary for this expression but there is internal redundancy within this region as two distinct upstream sequences of 18 and 22 bp respectively are sufficient for stage- and tissue-specific expression, albeit at reduced levels. A point mutation at -53 eliminates the ecdysone-mediated repression of the Sgs-3 promoter at pupariation. We report mosaicisms of expression within the salivary gland for a number of stably transformed lines.     

Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.     

Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.     

Photoregulated gene expression may involve ubiquitous DNA binding proteins.

Several promoter elements have previously been shown to influence the expression of the cab-E gene in Nicotiana plumbaginifolia. Here we demonstrate, by electrophoretic mobility shift and methylation interference assays, that a complex pattern of protein-DNA interactions characterizes this promoter. Among the multiple proteins identified, we focused on five different factors which either occupied important regulatory elements and/or were present in relatively large amounts in nuclear extracts. All of these proteins were distinguished on the basis of their recognition sequence and other biochemical parameters. One, GBF, interacted with a single sequence within the cab-E promoter homologous to the G-box found in many photoregulated and other plant promoters. A second factor, GA-1, bound to the GATA element which is located between the CAAT and TATA boxes of the cab-E and all other LHCII Type I CAB promoters. GA-1 also interacted in vitro with the I-boxes of the Arabidopsis rbcS-1A promoter and the as-2 site of the CaMV 35S promoter. Two other factors, GC-1 and AT-1, bound to multiple recognition sites localized within the GC-rich and AT-rich elements, respectively. GT-1, a protein which interacts with promoters of other light-regulated genes, bound to seven distinct sites distributed throughout the cab-E promoter.