Interspecific hybridization as a successful strategy to shed more lights into the regulation of proanthocyanidin biosynthesis and breed superior Lotus genotypes     

Project leaders

Francesco Paolocci, Oscar Adolfo Ruiz

Agreement

ARGENTINA - CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas

Call

CNR/CONICET 2013-2014

Department

Agriculture and Food

Thematic area

Biology, agriculture and food sciences

Status of the project

Extended

Report for renewal

joint-report2011-2012.pdf

Research proposal

Livestock is the world’s largest user of land resources (Steinfeld et al., 2006). Its productivity per hectare varies widely, largely depending on pastures. Pastures incorporating legumes are more productive and have higher nutritional value than legume-free ones (Sleugh et al., 2000). Improving the feeding value and environmental adaptability of legume pastures in marginal areas is becoming of paramount importance.
Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric and polymeric end-products of the flavonoid biosynthetic pathway that strongly affect plant quality traits (Tanner, 2004, Barry and McNabb, 1999). Whereas high concentrations of PAs in forage legumes depress voluntary feed intake, moderate quantities (about 5 mg PAs/g DW) prevents proteolysis during ensiling and rumen fermentation, thereby protecting ruminants against pasture bloat (Aerts et al., 1999; Li et al., 1996; Tanner et al., 1995) and decrease the amount of enteric methane produced -63% of total greenhouse gas (GHG) emission worldwide- (Beauchemin et al., 2010). PAs therefore make the conversion of plant proteins into animal proteins more efficient (Kingston-Smith and Thomas, 2003).
In the most valuable forage species, such as alfalfa (Medicago sativa) and clovers (Trifolium spp.), PAs accumulate in the seed coats only. Breeding programs aimed at engineering these pasture legumes to produce leaf PAs at agronomically important levels have been launched by different countries worldwide, however to date neither classical nor biotechnological approaches did succeed to this purpose (Paolocci et al. 2011). In the Lotus genus a high extent of variability for the accumulation of leaf PAs is present. This genus includes important forage legumes as L. corniculatus and L. tenuis. L. tenuis  is a diploid agronomically important species for the Argentinian Pampas (Soriano, 1991), where it is considered as a “keystone species”.  Yet all screened L. tenuis germplams accumulate only barely detectable levels of PAs (Escaray et al. 2012a). Conversely, L. corniculatus shows a significant degree of variation for the accumulation of PAs among its tetraploid genotypes along with a higher dry matter production than L tenuis (Escaray et al., 2012b).
Within the frame of CNR/CONICET bilateral project, the two Research Units (RU) have initially recovered and characterized a diploid, wild accession of L. corniculatus from an alkaline, salty and flooding susceptible area located in Albufera of Valencia (Spain), and then used it as pollen donor to cross plants from a commercial, highly productive L. tenuis variety looking to create a new gene assortment and different profiles of secondary metabolites within the Lotus genus by interspecific hybridization and output superior Lotus genotypes. Indeed, the vegetal material produced received the INNOVAR 2009 Award by MINCYT (Argentina) and allowed the proponents to select among the F2 population obtained by the cross of the original interspecific hybrids genotypes more tolerant than L. tenuis to alkaline and salty soils as well as to gain insight into the genetic control of PA biosynthesis in Lotus. To this purpose, quantitative and colorimetric analyses of PAs in different plant organs and tissues have been paralleled by the cloning and expression analysis by qRT-PCR of PA biosynthetic genes in the two parents, in their hybrids and progeny.
In summary, having shown as possible the use interspecific hybridization to transfer traits of agronomic interest within the Lotus genus, the tasks of the present project are: 1) the production of new Lotus interspecific hybrids and relative progeny to create new gene assortment for basic and applied purposes; 2) the dissection of the interplay between PA regulatory genes; 3) the assessment of the involvement of PAs in the symbiosis between Lotus spp and Rhizobia.
 
REFERENCES:
Aerts et al. (1999). Agric Ecosyst Environ 75:1-12.
Beauchemin et al. (2010). Agric Sys 103:371-379.
Bordeleau and Prévost (1994). Plant Soil 161:115-125.
Escaray et al. (2012). Plant Science 182:121-133.
Kingston-Smith and Thomas (2003). Ann Appl Biol 142:13-24.
Koes et al. (2005). Trends Plant Sci 10:236-242.
Li et al. (1996). J Sci Food Agric 70:89-101.
Pankhurst and Jones (1979). J Exp Bot 30:1095-1107.
Pankhurst et al. (1979). J Exp Bot 30:1085-1093.
Paolocci et al. (2005). J Exp Bot 56:1093-1103.
Paolocci et al. (2007). Plant Physiology 143:504-516.
Paolocci et al. (2011). J Exp Bot 62:1189-1189.
Sleugh et al. (2000). Agron J 92:24-29.
Steinfeld et al. (2006) Livestock’s long shadow: environmental issues and options, Food and Agricultural Organization, Rome, Italy.
Tanner (2004) Condensed tannins, in: K. Davies (Ed.), Blackwell Publishing Ltd, Oxford .
 
 

Research goals

1-Production of new Lotus interspecific hybrids and relative progeny to create additional gene assortment for basic and applied purposes:
New hybrids and relative progeny will be produced by crossing accessions/ecotypes of different Lotus species. To this end, accessions of the same ploidy level, already available in Argentinean laboratory through an international networking (EEC project LOTASSA), will be crossed. Exploring the possibility of producing fertile hybrid plants between different Lotus spp will also help us to glean more insights into the phylogeny within this genus and to test the possibility of transferring other agronomical important traits.
 
2-Dissection of the interplay between PA regulatory genes:
In all species analyzed thus far, the PA regulation appears to be mediated by the MYB-bHLH and WD-40 protein complex (Koes et al. 2005). Indeed, in Lotus spp analyses have shown that the contribution of the WD40 partner is dispensable, whereas a pivotal role is exerted by bHLH proteins and, more importantly, by the MYBs  that might act either as activators or repressors (Paolocci et al 2005; Robbins et al. 2005; Paolocci et al. 2007; Paolocci et al. 2011).
During the 2010-2012 bilateral project, we have cloned in L corniculatus full length cDNAs relative to TT2 and TT8 the MYB and bHLH gene, respectively, that activate PA pathway in different plant species. Sequencing coupled to qRT-PCR based experiments have shown that TT2 is organized as a small gene family in L. corniculatus and these members contribute differently to the overall accumulation of PAs in leaves and stems, with a member, named TT2b, that seems to play a major role. To get more insight into the role of TT2b and TT8 in the control of PAs, qRT-PCR analyses will be carried out in the F2 segregating population already available as well as in the new segregating population expected to be produced (Task 1).
 
3-Assessment of the involvement of PAs in the symbiosis of Lotus spp with Rhizobia:
Adaptability and production of legumes highly depend on the possibility for these plants to establish a functional symbiosis with Rhizobia (Escaray et al 2012a). Whether or not PAs play an active role in the process of mutual recognition between the two symbionts or in establishing active nodules is a long vexed question. Preliminary data obtained by the two RUs suggest that the capacity of forming active nodules is not independent from the levels and composition of root PAs. To gain more insight into this issue, a large screening will be pursued on roots from parental, hybrids and F2 populations with different Rhizobia species.
 

Last update: 30/04/2024