In this study, a total of 53 NLP genes were identified and sequentially named MsNLP1 to MsNLP53 on the basis of their chromosomal location in alfalfa. Previous studies have revealed nine NLP family genes in Arabidopsis, six in rice and nine in maize [3, 5, 11], indicating that different species have different numbers of NLPs. NLP family genes encode large proteins with slightly relatively low pI values in alfalfa (Table 1), consistent with results of other species, which indicates that these TFs are preferentially active under acidic conditions [5, 7, 25]. All MsNLPs identified in this study contain RWP-RK domains; however, only 23 members contain an intact PB1 domain, and the PB1 domain of the rest is partially deleted (Additional file 2), which was caused by sequence fragmentation during evolution or a sequencing error in the alfalfa genome[12]. Therefore, domain gain and loss are divergent forces that drive expansion of the NLP gene family [26, 27].
Phylogenetic analysis indicated that NLP genes can be divided into three groups in alfalfa (Fig. 1), which is consistent with previous evolutionary classifications in Arabidopsis, rice and maize. The number of MsNLP introns ranged from 1 to 10 in alfalfa, which was consistent with the results found in Arabidopsis [5], indicating that different species display similarities in terms of the diversity of the structure of NLP genes [5]. Previous research has shown that segmental duplication can result in a slower rate of intron gain than intron loss [28]. Group II had more introns than group I and group III(Fig. 2), indicating that group II was the most conserved because this group might contain the original genes. Segmental duplication events are critical for the rapid expansion and evolution of gene families and are essential for environmental adaptability and speciation [29–31]. According to our collinearity analysis of the MsNLP genes, we observed all pairs of four groups of fragment duplications (Fig. 3and Additional file 5), in which similar events have been found to have occurred in many species, including Arabidopsis thaliana, rice [5], tomato [32] and others. Therefore, the presence of duplication events implied that segmental duplications was the major evolutionary mechanisms that drove alfalfa NLP expansion in our research. If Ka/Ks > 1, a positive selection effect is considered, if Ka/Ks = 1, neutral selection is considered, and if Ka/Ks < 1, a purifying selection effect is considered [33]. Notably, the Ka/Ks ratio for all the NLP gene pairs except one (MsNLP05/08, which was greater than 1) was less than 1 (Fig. 4and Additional file 6), implicating mainly purifying selection.
Tissue-specific expression patterns of NLP genes in different tissues have been investigated in several plant species. For example, by assessing their expression levels in different tissues of Arabidopsis and rice, researchers have found that NLPs are expressed widely in almost all organs, including seeds, roots, stems, flowers, leaves, nodules, inflorescences, etc [7]. In Arabidopsis, AtNLP8 and AtNLP9 are highly expressed in senescing leaves and seeds compared with other organs, and their expression levels are moderate or very low. OsNLP1 and OsNLP3, whose transcripts constitute the majority of OsNLP transcripts, were found to be preferentially expressed in source organs [5]. It has also been reported that NLPs are highly expressed in the leaves, roots, male catkins, xylem, seeds, and female catkins of Populus trichocarpa [34, 35]. In the present study, the expression levels of most genes except MsNLP48 were higher in the leaves than in the roots and stems. MsNLP14/24/26/44/47/53 were highly expressed in young leaves, whereas MsNLP8/30/32 were highly expressed in mature leaves (Fig. 6), indicating that these genes may participate in leaf growth and development. In addition, we speculate that MsNLPs accumulate in leaves might be used for storing nitrogen to coordinate leaf expansion and photosynthetic capacity to promote leaf growth and biomass, according to previous reports in Brassica napus [34, 36]. Moreover, the expression of alfalfa NLPs in the roots was much lower than that in the leaf tissues (in which expression levels were high), indicating that NLPs are involved in mainly nitrate transport rather than the nitrate absorption process [11].
NLPs are essential TFs involved in nitrate signaling [37–39]. It has been reported that nitrate triggers nitrate-CPK (Ca2+-sensor protein kinase)-NLP signaling and nitrate-coupled CPK signaling to phosphorylate NLPs, which play a central role in nitrate signaling and integrate transcription, transport, metabolism and systematic growth processes in plants [14, 17, 40]. However, few NLP TFs involved in abiotic stress and phytohormone responses have been identified in alfalfa. Therefore, the goal of this study was to obtain further insights into the biological functions of MsNLP genes in response to abiotic stress and phytohormone treatment. Previous research has shown that abiotic stress not only affects alfalfa growth and development but also usually leads to decreases in crop yield and quality [41]. Our results revealed that all NLP genes were upregulated or downregulated under drought stress, especially at 12 h of prolonged stress(Fig. 7). Combined with the results of previous research that Arabidopsis thaliana AtNLP7 knockout plants were more drought resistant than wild-type plants, the results of the present study indicate that NLP TFs are related to drought resistance in plants. Although MsNLPs exhibited different expression patterns, MsNLPs were expressed in response to both salt and alkaline treatment. In addition, the expression of most MsNLP genes significantly decreased and increased following ABA treatment (Fig. 8). In Arabidopsis, AtNLP8 can directly bind to the promoter of an ABA catabolism-related enzyme-encoding gene to reduce ABA levels, which implies that NLP TFs may participate in the regulation of the ABA signaling pathway. Similarly, MsNLP genes were induced by GA and IAA phytohormone treatment. Overall, our study lays a foundation for further functional investigation of NLPs in alfalfa.