Calcium-dependent protein kinase (CDPK or CPK) and CDPK-related kinase (CRK) play an important role in plant growth, development, and adaptation to environmental stresses. However, their gene families had been yet inadequately investigated in Medicago truncatula. In this study, six MtCRK genes were computationally identified, they were classified into five groups with MtCDPKs based on phylogenetic relationships. Six pairs of segmental duplications were observed in MtCDPK and MtCRK genes and the Ka/Ks ratio, an indicator of selection pressure, was below 0.310, indicating that these gene pairs underwent strong purifying selection. Cis-acting elements of morphogenesis, multiple hormone responses, and abiotic stresses were predicted in the promoter region. The spatial expression of MtCDPKs and MtCRKs displays diversity. The expression of MtCDPKs and MtCRKs could be regulated by various stresses. MtCDPK4, 14, 16, 22, and MtCRK6 harbor both N-myristoylation site and palmitoylation site and were anchored on plasma membrane, while MtCDPK7, 9, and 15 contain no or only one N-acylation site and were distributed in cytosol and nucleus, suggesting that the N-terminal acylation sites play a key role in subcellular localization of MtCDPKs and MtCRKs. In summary, comprehensive characterization of MtCDPKs and MtCRKs provide a subset of candidate genes for further functional analysis and genetic improvement against drought, cold, salt and biotic stress.

Chinese milk vetch (Astragalus sinicus L.) is an important leguminous green manure that frequently suffers from seasonal drought. To improve its drought tolerance, the effects of rhizobia inoculation on drought tolerance and the underlying physiological mechanism were investigated. Drought tolerance in combination with nitrogen assimilation, free amino acids, and polyamines was investigated in milk vetch with active nodules (AN), with inactive nodules (LN), or without nodules (NN). AN plants had increased drought tolerance compared to LN and NN plants. Glutamine synthetase (GS), glutamine 2-oxoglutarate amino transferase (GOGAT), and glutamate dehydrogenase (GDH) activities were decreased after drought, with higher levels in AN plants than in LN and NN plants under both control and drought stress conditions. Higher levels of proline (Pro), arginine (Ala), alanine (Ala), and glutamate (Glu) were observed in AN plants compared with LN and NN plants. Putrescine (Put), spermidine (Spd), and spermine (Spm) levels were increased in response to drought, and higher levels of Put and Spd were maintained in AN plants. It is suggested that active nodulation leads to increased drought tolerance in milk vetch, which is associated with improved nitrogen fixation and ammonium assimilation, which in turn lead to the promotion of free amino-acid and polyamine synthesis.