Two genes encoding different truncated hemoglobins are regulated during root nodule and arbuscular mycorrhiza symbioses of Medicago truncatula

Abstract

The MtTrHb1 and MtTrHb2 genes of the model legume Medicago truncatula Gaertn. encode proteins homologous to truncated hemoglobins (TrHb) from plants and a range of different microorganisms. Induction of MtTrHb1 in root nodules and expression of MtTrHb2 in root nodules, as well as in mycorrhizal roots, were shown by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The promoters of both genes were PCR-amplified and fused to the gusAint coding region. By analysing these gusAint-fusions in transgenic root tissues, we were able to localize their activity in root nodules and in roots colonized by arbuscular mycorrhizal (AM) fungi. Whereas the promoter of MtTrHb1 was activated in the infected cells of the nitrogen-fixing zone of root nodules, the MtTrHb2 promoter was predominantly active in the nodule vascular tissue. This expression pattern correlates with the presence of an 'organ-specific element' (OSE)-like sequence in the MtTrHb1 promoter, which is not present in the MtTrHb2 regulatory unit. Concerning the AM symbiosis, only the MtTrHb2 promoter mediated an expression in arbuscule-containing cells and in the root vascular tissue of mycorrhizal root segments colonized by the fungus Glomus intraradices.

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

“…Samples were also treated with TURBO™ DNase (Ambion) according to the manufacturer's instructions. The RNA samples were routinely checked for DNA contamination by reverse transcription polymerase chain reaction (RT-PCR) analysis, using primers MtTef-f 5 ′ -AAGCTAGGAGGTATTGACAAG-3 ′ and MtTef-r 5 ′ -ACTGTGCAGTAGTACTTGGTG-3′ for MtTefa (Vieweg et al 2005) and the One-…”

“…The primer pairs used were MtTef-f and MtTef-r 5′ for MtTefa (Vieweg et al 2005); NRf 5 ′ -CTTTAAACCCGCGTGAGAAA-3 ′ and NRr 5 ′ -TCATCTACGCTGCTTGTTGG-3′ for NR (accession number AJ621867; Weidmann et al 2004); and NiRf 5 ′ -TATGTTGGAATTCACGTTCCAG-3 ′ and NiRr 5 ′ -TGCCACAAGTGTTTTCATTAGG-3 ′ for NiR (accession number AJ621861; Weidmann et al 2004). Real-time RT-PCR was performed on the StepOne Real-Time PCR System (Applied Biosystems) using the following program: 10 min preincubation at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C.…”

Nodules and oxygen

“…The third class of plant Hbs is represented by homologues of microbial truncated Hbs (2-on-2 Hbs; reviewed by Wittenberg et al 2002) which seem to have evolved independently of class I and class II Hbs (Garrocho-Villegas et al 2007). Their function has been suggested to be linked to NO scavenging as well (Milani et al 2003;Vieweg et al 2005), but proof has yet to be provided. Truncated hemoglobins are formed in infected cells of legume nodules (Vieweg et al 2005).…”

“…Their function has been suggested to be linked to NO scavenging as well (Milani et al 2003;Vieweg et al 2005), but proof has yet to be provided. Truncated hemoglobins are formed in infected cells of legume nodules (Vieweg et al 2005).…”

“…In the legume M. truncatula, two trHb genes were found to be expressed at elevated levels in nodules compared to roots, one of them specifically in infected cells (Vieweg et al 2005). Expression of plant trHb genes in nodules could also be shown in other legume species (Phaseolus vulgaris, GenBank accession CV537139; soybean, GenBank accession no.…”