Jean-Christophe Breitler

Molecular genetics of rice root development

Plant roots have a large range of functions, including acquisition of water and nutrients, as well as structural support. Dissecting the genetic and molecular mechanisms controlling rice root development is critical for the development of new rice ideotypes that are better adapted to adverse conditions and for the production of sustainably achieved rice yield potential. Most knowledge regarding the gene networks involved in root development has been accumulated in the model dicotyledon plant species Arabidopsis thaliana. Rice, the model monocotyledon species, presents several singularities compared to A. thaliana, including a root architecture characterized by a fibrous root system comprising five types of embryonic and postembryonic roots. The anatomy and morphology of the rice root system, which is typical for a cereal, differs from that of A. thaliana, for instance, by the presence of a lysigenous cortex and additional cell layers compared to the dicotyledon model. Moreover, the structure and functions of the root apical meristem (RAM) of rice are distinct from those of A. thaliana. Recently, several rice root mutants have been identified via forward or reverse genetics, and these will aid in forming hypothesis to characterize either the divergence or conservation of genetic pathways relative to A. thaliana. Furthermore, these mutants will help to identify key genes in rice roots that may be missing in A. thaliana. This review summarizes both classical and recent data concerning the molecular genetics of rice root development, including root anatomy and morphology, RAM structure, RAM patterning, and root mutants.

A Novel Two T-DNA Binary Vector Allows Efficient Generation of Marker-free Transgenic Plants in Three Elite Cultivars of Rice (Oryza sativa L.)

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82–90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

Expression of a Bacillus thuringiensis cry1B synthetic gene protects Mediterranean rice against the striped stem borer.

Bacillus thuringiensis Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01% to 0.4% of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90–100% mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4% of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations.

Modulating Rice Stress Tolerance by Transcription Factors

Abstract Plants are non-mobile organisms and have to adapt to environmental stresses mostly by modulating their growth and development in addition to physiological and biochemical changes. Transcription factors (TFs) regulate genome expression in response to environmental and physiological signals, and some of them switch on plant adaptive developmental and physiological pathways. One TF is encoded by a single gene but regulates the expression of several other genes leading to the activation of complex adaptive mechanisms and hence represents major molecular targets to genetically improve the tolerance of crop plants against different stresses. In this review an updated account of the discovery of TFs involved in biotic and abiotic stress tolerance in the model monocotyledonous plant, rice (Oryza sativa L.) is presented. We illustrate how the elucidation of the function of these TFs can be used to set up genetic engineering strategies and to rationalize molecular breeding using molecular assisted selection towards enhancement of rice tolerance to various stresses. Attempts have also been made to provide information on the molecular mechanisms involved in Coudert is granted by the French Ministry of Scientific Research, Dr. P. K. Pati was granted by an Indian Boycast fellowship, the research is supported by University Montpellier 2 and CIRAD.

Coffee (Coffea arabica L.).

Coffee (Coffea sp.) is a perennial plant widely cultivated in many tropical countries. It is a cash crop for millions of small farmers in these areas. As for other tree species, coffee has long breeding cycles, which makes conventional breeding programs time-consuming. For that matter, genetic transformation can be an effective way to introduce a desired trait in elite varieties or for functional genomics. In this chapter, we describe two highly efficient and reliable Agrobacterium-mediated transformation techniques developed for the C. arabica cultivated species: (1) A. tumefaciens to study and introduce genes conferring resistance/tolerance to biotic (coffee leaf rust, insects) and abiotic stress (drought, heat, seed desiccation) in fully transformed plants and (2) A. rhizogenes to study candidate gene expression for nematode resistance in transformed roots.

Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit

Fine-tuning of DREB1D expression in stomatal guard cells under water deficit is mediated differentially by promoter haplotypes from sensitive and tolerant coffee genotypes.

Healthy Tropical Plants to Mitigate the Impact of Climate Change—As Exemplified in Coffee

The impacts of climate change on coffee trees are hard to foresee and dependent on the cropping system (ranging from high input monocultures to almost natural agroforestry associations), soil and water resources. Pests and diseases are also affected. Research on adaptation to climate change is mainly focused on cereals but there are few studies on perennial tropical crops where stress can alter the behaviour of the plant for several seasons. Some parasites can adapt more quickly to climate change than the perennial host plants and spread into new habitats. The aim of CIRAD’s coffee genetic improvement research is to obtain ‘healthy’ crops, i.e. more resilient and resistant. Here we present integrative approaches focused on the functioning of the genome (transcriptomic, metabolomic, genomic, epigenetic) and the whole plant, as well as adaptive responses to combined stresses. The goal is to propose targets for coffee breeding in order to improve resistance to coffee rust, nematode control and adaptation to drought.

A single-step method for RNA isolation from tropical crops in the field

The RNAzol RT reagent was used to provide pure RNA from human cells. We develop a protocol using RNAzol RT reagent to extract pure RNA from plants tissues and demonstrate that this RNA extraction method works not only at room temperature but also at elevated temperatures and provides the simplest and most effective single-step method to extract pure and undegraded RNA directly from tropical plants in the field. RNA extraction directly in a complex field environment opens up the way for studying gene-environment interactions at transcriptome level to decipher the complex regulatory network involved in multiple-stress responses.

The mariner Mos1 transposase produced in tobacco is active in vitro.

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.

Juvenile Coffee Leaves Acclimated to Low Light Are Unable to Cope with a Moderate Light Increase

The understorey origin of coffee trees and the strong plasticity of Coffea arabica leaves in relation to contrasting light environments have been largely shown. The adaptability of coffee leaves to changes in light was tested under controlled conditions by increasing the illumination rate on C. arabica var. Naryelis seedlings acclimated to low light conditions and observing leaf responses at three different developmental stages (juvenile, growing and mature). Only mature leaves proved capable of adapting to new light conditions. In these leaves, different major mechanisms were found to contribute to maintaining a good photosynthetic level. With increased illumination, a high photosynthetic response was conserved thanks to fast nitrogen remobilization, as indicated by SPAD values and the photorespiration rate. Efficient photoprotection was accompanied by a great ability to export sucrose, which prevented excessive inhibition of the Calvin cycle by hexose accumulation. In contrast, in younger leaves, increased illumination caused photodamage, observable even after 9 days of treatment. One major finding was that young coffee leaves rely on the accumulation of chlorogenic acids, powerful antioxidant phenolic compounds, to deal with the accumulation of reactive oxygen species rather than on antioxidant enzymes. Due to a lack of efficient photoprotection, a poor ability to export sucrose and inadequate antioxidant protection, younger leaves seemed to be unable to cope with increased illumination. In these leaves, an absence of induced antioxidant enzyme activity was accompanied, in growing leaves, by an absence of antioxidant synthesis or, in juvenile leaves, inefficient synthesis of flavonoids because located in some epidermis cells. These observations showed that coffee leaves, at the beginning of their development, are not equipped to withstand quick switches to higher light levels. Our results confirm that coffee trees, even selected for full sunlight conditions, remain shade plants possessing leaves able to adapt to higher light levels only when mature.