Study of nsLTPs in Lotus japonicus genome reveal a specific epidermal cell member (LjLTP10) regulated by drought stress in aerial organs with a putative role in cutin formation.

The cuticle is the first defense against pathogens and the second way water is lost in plants. Hydrophobic layers covering aerial plant organs from primary stages of development form cuticle, including major classes of aliphatic wax components and cutin. Extensive research has been conducted to understand cuticle formation mechanisms in plants. However, many questions remain unresolved in the transport of lipid components to form cuticle. Database studies of the Lotus japonicus genome have revealed the presence of 24 sequences classified as putative non-specific lipid transfer proteins (nsLTPs), which were classified in seven groups; four groups were selected because of their expression in aerial organs. LjLTP8 forms a cluster with DIR1 in Arabidopsis thaliana while LjLTP6, LjLTP9, and LjLTP10 were grouped as type I LTPs. In silico studies showed a high level of structural conservation, and substrate affinity studies revealed palmitoyl-CoA as the most likely ligand for these LTPs, although the Lyso-Myristoyl Phosphatidyl Choline, Lyso-myristoyl phosphatidyl glycerol, and Lyso-stearyl phosphatidyl choline ligands also showed a high affinity with the proteins. The LjLTP6 and LjLTP10 genes were expressed in both the stems and the leaves under normal conditions and were highly induced during drought stress. LjLTP10 was the most induced gene in shoots during drought. The gene was only expressed in the epidermal cells of stems, primordial leaves, and young leaflets. LjLTP10 was positively regulated by MeJA but repressed by abscisic acid (ABA), ethylene, and H2O2, while LjLTP6 was weakly induced by MeJA, repressed by H2O2, and not affected by ABA and ethylene. We suggest that LjLTP10 is involved in plant development of stem and leaf cuticle, but also in acclimation to tolerate drought stress in L. japonicus.

Molecular characterisation of Ltchi7, a gene encoding a Class III endochitinase induced by drought stress in Lotus spp.

Chitinases are enzymes that digest chitin molecules, present principally in insects and fungi. In plants, these enzymes play an important role in defence against pathogen attack, although they have also been described as induced by mechanical damage, ozone, heavy metals, cold, salinity, etc. Using an annealing control primer, we isolated a gene fragment whose translated sequence has high homology with a class III endochitinase. The gene, named Ltchi7, consisted of one ORF of 1005 bp, which codes for a peptide of 334 amino acids, including a deduced signal peptide of 27 amino acid that directs protein to the extracellular space. Phylogenetic analysis suggests that Ltchi7 is within a cluster that includes Sesbania rostrata, Medicago sativa and Glycine max class III endochitinases. This group is differentiated from other species of endochitinases by the presence of an additional extension in carboxy-terminal region. Moreover, in comparison with the majority of chitinases, Ltchi7 has two additional cysteine residues, which, according to 3D modelling studies, are very close. Gene expression analysis showed enhanced transcript abundance of this gene during drought stress in Lotus tenuis and Lotus japonicus, compared with growth under normal conditions. Furthermore, its expression is restricted to nodules and roots. Expression of this gene was also induced by salt stress, hydrogen peroxide and weakly with abscisic acid.