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MADS-box genes are important transcription factors in eukaryotes that are involved in regulating the growth and development of many plants. Jiaobai is a special aquatic vegetable formed through the stem expansion of Zizania latifolia infected by Ustilago esculenta and is wildly cultivated in Southeast Asia. To date, the biological function of MADS-box genes in Z. latifolia remains largely unknown. In this study, a genome-wide search for MADS-box genes in Z. latifolia was performed, and 87 MADS-box genes were identified. According to the phylogenetic analysis, there were 27 type I genes and 60 type II genes. The type I genes were mainly distributed in the Mα, Mβ and Mγ subfamilies, and the type II genes were divided into 14 subfamilies and unevenly distributed across 17 chromosomes. The encoded protein sequences ranged from 91 to 433 amino acids, the number of exons was 1~14, and most type I genes did not contain introns. The promoter region contained a large number of functional domains related to light response, plant hormones, stress and the meristem. An analysis of the expression patterns showed that there were massive differences in the expression of the MADS-box genes in different tissues, with six genes highly expressed in leaves and eight genes highly expressed in the stem apical meristem. Photoperiod and temperature were found to regulate the formation of the stem gall (SG) of Z. latifolia, and short-day (SD) conditions had an obvious promoting effect. ZlMADS57 and ZlMADS85 were significantly increased in SG under SD. Further analysis of the expression levels of MADS-box genes during the developmental stages of Z. latifolia found that the ZlMADS45, ZlMADS57, ZlMADS81 and ZlMADS85 were highly expressed 10 days before the SG formation, and ZlMADS66 reached its highest value on the day when the SG began forming. ZlMADS14, ZlMADS15, ZlMADS32, ZlMADS36, ZlMADS59 and ZlMADS71 were highly expressed after SG expansion, indicating that the MADS-box gene may play an important role in regulating SG formation and the subsequent expansion of Jiaobai. This study provides a reference for the functional identification of the MADS-box gene family and establishes a foundation for regulating the maturity of Z. latifolia.

Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance -catenin expression and activity, and mechanical unloading decreased -catenin expression through MACF1. Moreover, -catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing -catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. Refereed/Peer-reviewed

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1 knockdown increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. This article is protected by copyright. All rights reserved