Raffaella Greco

Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA–insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Activation Tagging Using the En-I Maize Transposon System in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays)En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant calledthread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that theEn-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.

EU-OSTID: A collection of transposon insertional mutants for functional genomics in rice

A collection of 1373 unique flanking sequence tags (FSTs), generated from Ac/Ds and Ac transposon lines for reverse genetics studies, were produced in japonica and indica rice, respectively. The Ds and Ac FSTs together with the original T-DNAs were assigned a position in the rice genome sequence represented as assembled pseudomolecules, and found to be distributed evenly over the entire rice genome with a distinct bias for predicted gene-rich regions. The bias of the Ds and Ac transposon inserts for genes was exemplified by the presence of 59% of the inserts in genes annotated on the rice chromosomes and 41% present in genes transcribed as disclosed by their homology to cDNA clones. In a screen for inserts in a set of 75 well annotated transcription factors, including homeobox-containing genes, we found six Ac/Ds inserts. This high frequency of Ds and Ac inserts in genes suggests that saturated knockout mutagenesis in rice using this strategy will be efficient and possible with a lower number of inserts than expected. These FSTs and the corresponding plant lines are publicly available through OrygenesDB database and from the EU consortium members.

BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways.

The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves.

Immunogenicity and tolerance following HIV-1/HBV plant-based oral vaccine administration

Transgenic tobacco plants expressing a HIV-1 polyepitope associated with hepatitis B (HBV) virus-like particles (VLPs) were previously described. It is demonstrated here that oral administration of these transgenic plants to humanized HSB mice to boost DNA-priming can elicit anti-HIV-1 specific CD8+ T cell activation detectable in mesenteric lymph nodes. Nevertheless, a significant regulatory T cell activation was induced in vivo by the vaccination protocols. The balance between tolerance and immunogenicity remains the main concern in the proof of concept of plant-based vaccine.

Genome-Wide Association Study for Traits Related to Plant and Grain Morphology, and Root Architecture in Temperate Rice Accessions

Background In this study we carried out a genome-wide association analysis for plant and grain morphology and root architecture in a unique panel of temperate rice accessions adapted to European pedo-climatic conditions. This is the first study to assess the association of selected phenotypic traits to specific genomic regions in the narrow genetic pool of temperate japonica. A set of 391 rice accessions were GBS-genotyped yielding—after data editing—57000 polymorphic and informative SNPS, among which 54% were in genic regions. Results In total, 42 significant genotype-phenotype associations were detected: 21 for plant morphology traits, 11 for grain quality traits, 10 for root architecture traits. The FDR of detected associations ranged from 3 · 10−7 to 0.92 (median: 0.25). In most cases, the significant detected associations co-localised with QTLs and candidate genes controlling the phenotypic variation of single or multiple traits. The most significant associations were those for flag leaf width on chromosome 4 (FDR = 3 · 10−7) and for plant height on chromosome 6 (FDR = 0.011). Conclusions We demonstrate the effectiveness and resolution of the developed platform for high-throughput phenotyping, genotyping and GWAS in detecting major QTLs for relevant traits in rice. We identified strong associations that may be used for selection in temperate irrigated rice breeding: e.g. associations for flag leaf width, plant height, root volume and length, grain length, grain width and their ratio. Our findings pave the way to successfully exploit the narrow genetic pool of European temperate rice and to pinpoint the most relevant genetic components contributing to the adaptability and high yield of this germplasm. The generated data could be of direct use in genomic-assisted breeding strategies.

The NTT transcription factor promotes replum development in Arabidopsis fruits.

Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary. In this work we describe the role for the zinc-finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss of function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1 and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.

Altered expression of the bZIP transcription factor DRINK ME affects growth and reproductive development in Arabidopsis thaliana

Here we describe an uncharacterized gene that negatively influences Arabidopsis growth and reproductive development. DRINK ME (DKM; bZIP30) is a member of the bZIP transcription factor family, and is expressed in meristematic tissues such as the inflorescence meristem (IM), floral meristem (FM), and carpel margin meristem (CMM). Altered DKM expression affects meristematic tissues and reproductive organ development, including the gynoecium, which is the female reproductive structure and is determinant for fertility and sexual reproduction. A microarray analysis indicates that DKM overexpression affects the expression of cell cycle, cell wall, organ initiation, cell elongation, hormone homeostasis, and meristem activity genes. Furthermore, DKM can interact in yeast and in planta with proteins involved in shoot apical meristem maintenance such as WUSCHEL, KNAT1/BP, KNAT2 and JAIBA, and with proteins involved in medial tissue development in the gynoecium such as HECATE, BELL1 and NGATHA1. Taken together, our results highlight the relevance of DKM as a negative modulator of Arabidopsis growth and reproductive development.