Rice Phytochrome Is Biologically Active in Transgenic Tobacco

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.     

The 5''-proximal region of the wheat Cab-1 gene contains a 268-bp enhancer-like sequence for phytochrome response.

We have previously reported that the expression of the wheat Cab-1 gene is subject to phytochrome regulation and a 1.8-kb 5' upstream sequence of this gene is sufficient for the regulated expression. To delineate sequences for the phytochrome response we analyzed a series of 5' deletion mutants as well as chimeric gene constructs comprising different sequences of the Cab-1 upstream region in transgenic tobacco seedlings. We found that a deletion mutant containing a 357-bp 5' upstream sequence still exhibits maximal levels of phytochrome-regulated expression. A 268-bp enhancer-like element, located between -89 and -357, is responsible for the phytochrome response of the Cab-1 gene; sequences upstream from -357 to -843 and downstream from -124 to +1100 are probably not involved. Finally, we show that the Cab-1 mRNA stability is not regulated by phytochrome.     

Arabidopsis rbcS genes are differentially regulated by light.

Individual members of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene family are differentially regulated by light of different qualities. In 10-d-old etiolated seedlings, the expression of only three of the four genes is under inductive phytochrome control. rbcS mRNA levels reach a maximum (3- to 5-fold higher than the dark level) about 6 h after a red light pulse, but the rate of decay differs among the genes. Moreover, rbcS 2B requires a higher fluence for induction. At early stages of development, rbcS 1A, 2B, and 3B are highly expressed in the dark and cannot be further induced by red light, indicating a developmental component in the overall regulatory mechanism. Continuous light experiments indicate that high-irradiance responses may play a role in the induction of at least three of the four rbcS genes. Under conditions of phytochrome saturation, rbcS 1A is insensitive to blue light pulses, whereas among the three B locus genes, at least rbcS 3B appears to respond to a blue-light photoreceptor. These results add to the data suggesting that individual members of rbcS gene families in higher plants may be subject to a variety of differing regulatory mechanisms.     

Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.

Expression of rbcS genes encoding small subunit of rubisco, most abundant protein in green tissue, is regulated by at least three parameters--tissue type, light conditions and stage of development. One of the green tissue-specific promoters of rbcS gene family was isolated from pigeonpea by PCR. Expression of uidA gene encoding beta-glucuronidase in the transgenic tobacco plants under the control of pigeonpea rbcS promoter, clearly showed that this promoter was as strong as pea rbcS3A promoter characterized earlier. Study of the sequence similarity with pea rbcS3A promoter, especially the region (boxes I and III) that is required for rbcS3A expression, showed more than 50% divergence. In contrast, pigeonpea promoter sequence isolated in the present study was more similar to that of spinach and rice rbcS promoters.     

转录后基因沉默系统研究烟草rbcS基因功能

初步建立了利用病毒载体诱导转录后基因沉默系统研究烟草(Nicotiana benthamiana)1,5-二磷酸核酮糖羧化酶／加氧酶小亚基(Ribulose-1,5-bisphosphate carboxylase／oxylase small subunit,rbcS)基因功能的模式。用携带与1,5-二磷酸核酮糖羧化酶／加氧酶小亚基基因同源的cDNA片段的烟草脆裂病毒载体(pTV．rbcS)侵染烟草(Nicotiana benthamiana),诱导内源rbcS基因沉默并在此基础上建立了研究rbcS基因功能的模式：初步进行了rbcS基因沉默后的表型分析、转录水平分析、蛋白质表达水平分析以及利用HPLC方法定量分析rbcS基因沉默后的光合色素变化。结果表明：病毒诱导基因沉默瞬时表达体系中烟草最佳侵染时期为苗龄21-24d,用于侵染的重组农杆菌的最佳浓度的OD值为1～1．5;烟草Rubisco小亚基的表达量可能调节Rubisco大亚基的表达量;烟草rbcS基因与光合作用中的光能收集无关。对rbcS基因沉默的烟草叶片及对照烟草叶片的部分重要光合作用指标分析表明,运用烟草脆裂病毒载体诱导转录后基因沉默系统研究烟草rbcS基因功能具有可行性,为进一步深入研究rbcS基因功能奠定了基础。     

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.     

Rice phytochrome A controls apical hook opening after a single light pulse in transgenic tobacco seedlings

Apical hook opening in tobacco seedlings can be induced by a single red light pulse and this induction can be reverted by a subsequent far-red light pulse. The slow hook opening kinetics and the reversibility of an inductive light pulse even after 8 h of darkness indicate the involvement of stable phytochrome. Compared with wild-type, transgenic BN1 seedlings which overexpress rice phytochrome A exhibit a higher sensitivity to low irradiance red light pulses. Moreover, in BN1 seedlings an inductive red light pulse is only partially reversible even after 30 min, whereas wild-type tobacco seedlings show complete reversibility during the entire hook opening process. The data found show that rice phytochrome A is active in transgenic tobacco seedlings in controlling hook opening and that the introduced rice phytochrome A and the endogenous stable phytochrome behave differently in this response.     

Sequences 5'' to translation start regulate expression of petunia rbcS genes.

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes.     

shygrl1 is a mutant affected in multiple aspects of photomorphogenesis

We have used a counter-selection strategy based on aberrant phytochrome regulation of an Lhcb gene to isolate an Arabidopsis mutant designated shygrl1 (shg1). shg1 seedlings have reduced phytochrome-mediated induction of the Lhcb gene family, but normal phytochrome-mediated induction of several other genes, including the rbcS1a gene. Additional phenotypes observed in shg1 plants include reduced chlorophyll in leaves and additional photomorphogenic abnormalities when the seedlings are grown on medium containing sucrose. Mutations in the TATA-proximal region of the Lhcb1*3 promoter that are known to be important for phytochrome regulation affected reporter gene expression in a manner similar to the shg1 mutation. Our results are consistent with the possibility that the mutation either leads to defective chloroplast development or to aberrant phytochrome regulation. They also add to the evidence of complex interactions between light- and sucrose-regulated pathways.