Overexpression of Medicago MtCDFd1_1 Causes Delayed Flowering in Medicago via Repression of MtFTa1 but Not MtCO-Like Genes

Abstract

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

The Candidate Photoperiod Gene MtFE Promotes Growth and Flowering in Medicago truncatula

“…However, in this mutant the delay in flowering is only moderate and could be explained by the reduction in MtFTa1 expression, suggesting that the disruption of both the MtFTb genes had little effect on flowering. This is consistent with previous flowering time observations when overexpression of MtCDFd1_1 also disrupted expression of both MtFTb genes, but the late flowering observed was dependent on loss of MtFTa1 function (Zhang et al, 2019).…”

“…Furthermore, the expression of FT-like genes are significantly reduced in LATE1 ( Psgi ) mutants ( Hecht et al, 2007 , 2011 ) and a dominant late flowering pea CDF mutant was recently shown to act in the same pathway ( Ridge et al, 2016 ). Similarly, in M. truncatula overexpression of the MtCDFd1_1 gene resulted in late flowering relative to wild type plants in LD photoperiods resulting in the plants flowering in a day-neutral manner ( Zhang et al, 2019 ). In addition, in both pea and M. truncatula , homologs of PHYA strongly promote flowering in LD with Mtphya mutants having strongly reduced levels of the LD-induced FT-like genes ( Weller et al, 2001 , 2004 ; Jaudal et al, 2020 ).…”

A novel Medicago truncatula calmodulin‐like protein (MtCML42) regulates cold tolerance and flowering time

“…The expression of MtCML42 was induced by cold but was reduced by ABA treatment, indicating the coldinduced MtCML42 expression was not associated with ABA. Among the large number of CMLs found in plant species a few CMLs are known to be expressed in response to cold, such as AtCML24 (Delk et al, 2005), CsCML16, CsCML18-2 and CsCML42 (Ma et al, 2019), ShCML44 (Munir et al, 2016) and MtCML40 (Zhang, Jiang, et al, 2019). The role of MtCML42 in the regulation of cold tolerance was documented in the present study.…”

“…The results suggest that MtCML42 positively regulates cold tolerance. A few CMLs, such as AtCML10 (Cho et al, 2016), AtCML37 (Scholz et al, 2015) and ShCML44 (Munir et al, 2016), positively regulate cold, drought and salinity tolerance, whereas other CMLs, such as AtCML9 (Magnan et al, 2008), AtCML20 (Wu et al, 2017), AtCML42 (Vadassery et al, 2012 and MtCML40 (Zhang, Jiang, et al, 2019), negatively regulate abiotic stress tolerance. The positive or negative regulation of CMLs on abiotic stress tolerance is associated with jasmonic acid (Scholz et al, 2015;Vadassery et al, 2012;Zhu, Robe, et al, 2017) or ABA signaling (Delk et al, 2005;Magnan et al, 2008;Wu et al, 2017).…”

“…In addition, CYCLING DOF FACTOR d1_1 (MtCDFd1_1) delayed flowering in M. truncatula (Zhang, Wang, et al, 2019), whereas PHYTOCHROME A (MtPHYA) and PHLOEM-SPECIFIC MYB-RELATED GENE (MtFE) promotes flowering in M. truncatula (Jaudal et al, 2020;Thomson et al, 2021). Overexpression of MtSVP genes in Arabidopsis leads to delayed flowering (Jaudal et al, 2014), whereas MtCDFd1_1 negatively regulates photoperiodic flowering via the repression of MtFTa1 expression (Zhang, Jiang, et al, 2019). MtPHYA promotes early flowering by regulating the expression of LD-induced flowering time clockrelated genes (Jaudal et al, 2020).…”