Leaf senescence is a complex organized developmental stage limiting the yield of crop plants, and alfalfa is an important forage crop worldwide. However, our understanding of the molecular mechanism of leaf senescence and its influence on biomass in alfalfa is still limited. In this study, RNA sequencing was utilized to identify differentially expressed genes (DEGs) in young, mature, and senescent leaves, and the functions of key genes related to leaf senescence. A total of 163,511 transcripts

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... nd 77,901 unigenes were identified from the transcriptome, and 5,133 unigenes were differentially expressed. KEGG enrichment analyses revealed that ribosome and phenylpropanoid biosynthesis pathways, and starch and sucrose metabolism pathways are involved in leaf development and senescence in alfalfa. GO enrichment analyses exhibited that six clusters of DEGs are involved in leaf morphogenesis, leaf development, leaf formation, regulation of leaf development, leaf senescence and negative regulation of the leaf senescence biological process. The WRKY and NAC families of genes mainly consist of transcription factors that are involved in the leaf senescence process. Our results offer a novel interpretation of the molecular mechanisms of leaf senescence in alfalfa. PeerJ reviewing PDF | ABSTRACT 11 Leaf senescence is a complex organized developmental stage limiting the yield of crop plants, 12 and alfalfa is an important forage crop worldwide. However, our understanding of the molecular 13 mechanism of leaf senescence and its influence on biomass in alfalfa is still limited. In this study, 14 RNA sequencing was utilized to identify differentially expressed genes (DEGs) in young, mature, 15 and senescent leaves, and the functions of key genes related to leaf senescence. A total of 16 163,511 transcripts and 77,901 unigenes were identified from the transcriptome, and 5,133 17 unigenes were differentially expressed. KEGG enrichment analyses revealed that ribosome and 18 phenylpropanoid biosynthesis pathways, and starch and sucrose metabolism pathways are 19 involved in leaf development and senescence in alfalfa. GO enrichment analyses exhibited that 20 six clusters of DEGs are involved in leaf morphogenesis, leaf development, leaf formation, 21 regulation of leaf development, leaf senescence and negative regulation of the leaf senescence 22 biological process. The WRKY and NAC families of genes mainly consist of transcription 23 factors that are involved in the leaf senescence process. Our results offer a novel interpretation of 24 the molecular mechanisms of leaf senescence in alfalfa. 25 26 INTRODUCTION 27 Leaves are key photosynthetic organs that produce carbohydrates that provide energy for plant 28 development. In the last stage of leaf development, the photosynthate-producing ability of the PeerJ reviewing PDF | Manuscript to be reviewed 29 leaf is weakened, and the leaf enters the senescence stage (Quirino et al., 2000). During its life 30 span, the leaf undergoes three main developmental phases: initial leaves, maturation, and 31 senescence (Buchanan-Wollaston, 1997). Much more nutrient input is initially needed to support 32 leaf growth and development. The mature leaves are mainly responsible for providing a source 33 of carbon, and this function continues until the onset of senescence. During leaf senescence, the 34 nutrients in leaves, including nitrogen, metals, and phosphorus, are reallocated to other tissues 35 and organs to support plant development. Moreover, the leaf cells undergo significant changes in 36 their cellular metabolism and cellular structures are degenerated (Rattan and Hayflick, 2016;