Characterization of the Ferredoxin-Gogat gene (OsGog2 clone) expression in rice

Ferredoxin-dependent glutamate synthase (Fd-Gogat; EC 1.4.7.1) in leaf and root plastids is the last enzyme involved in the pathway of nitrate assimilation in higher plants. Arabidopsis thaliana expresses two different genes: the first, light regulated, specific of green tissues and the second expressed in other tissues. In this work, we investigated whether in our clone, OsGog2 AC Y12595, this gene is up-regulated by light or it is expressed under darkness. Fd-Gogat specific activity, protein and mRNA increased after light treatment in rice shoots. In roots, the activity and the protein content remained constant, whereas the mRNA is repressed by light treatment. The results obtained using a specific probe, situated in the 3′ untranslated region of the OsGog2 cDNA, indicated that OsGog2 gene is up-regulated by light and that its expression is tissue specific and suggested that a dark expressed Fd-Gogat gene could be present in rice similarly as in Arabidopsis.     

Molecular Cloning of PbSTKL1 Gene from Plasmodiophora brassicae Expressed during Clubroot Development

Infection of crucifers by the obligate plant pathogen Plasmodiophora brassicae Woron. results in the formation of clubroot disease in these plants. Plasmodiophora brassicae gene expression during disease development was studied by differential display analysis of total RNA extracted from the roots of Chinese cabbage inoculated with the pathogen. In a series of experiments, 30 differentially expressed bands of cDNA were detected, and the expression of clone no. 17 was confirmed in clubbed roots. Southern blot analysis showed that this clone was a single‐copy gene in the P. brassicae genome. Putative amino acid sequence analysis of the full‐length cDNA of clone no. 17 (4.6 kb, designated PbSTKL1) revealed a serine/threonine kinase‐like domain at the C‐terminal region and a coiled‐coil structure in the middle region of the putative protein. PbSTKL1 expression increased strongly beginning 30 days after inoculation and was coincident with resting spore formation.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

Circadian clock component,LHY, tells a plant when to respond photosynthetically to light in nature

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY (irLHY) and NaTOC1 (irTOC1). We characterized growth and light‐ and dark‐adapted photosynthetic rates (A_c) throughout a 24 h day in empty vector‐transformed (EV), irLHY, and irTOC1 plants in the field, and in NaPhyA‐ and NaPhyB1‐silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced A_c earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field. irLHY, but not EV plants, responded to light in the night by rapidly increasing A_c. Under controlled conditions, EV plants rapidly increased A_c in the day compared to dark‐adapted plants at night; irLHY plants lost these time‐dependent responses. The role of NaLHY in gating photosynthesis is independent of the light‐dependent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light‐mediated in native tobacco.     

Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus

Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napuscDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.