The two novel CCCH zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, play important roles in regulating flowering and senescence redundantly in Arabidopsis. The CCCH zinc-finger proteins and K-homolog (KH) proteins play important roles in plant development and stress responses. However, the biological functions of many CCCH zinc-finger proteins and KH proteins remain uncharacterized. In Arabidopsis, KHZ1 and KHZ2 are characterized as two novel CCCH zinc-finger and KH domain proteins which belong to subfamily VII in CCCH family. We obtained khz1, khz2 mutants and khz1 khz2 double mutants, as well as overexpression (OE) lines of KHZ1 and KHZ2. Compared with the wild type (WT), the khz2 mutants displayed no defects in growth and development, and the khz1 mutants were slightly late flowering, whereas the khz1 khz2 double mutants showed a pronounced late flowering phenotype. In contrast, artificially overexpressing KHZ1 and KHZ2 led to the early flowering. Consistent with the late flowering phenotype, the expression of flowering repressor gene FLC was up-regulated, while the expression of flowering integrator and floral meristem identity (FMI) genes were down-regulated significantly in khz1 khz2. In addition, we also observed that the OE plants of KHZ1 and KHZ2 showed early leaf senescence significantly, whereas the khz1 khz2 double mutants showed delayed senescence of leaf and the whole plant. Both KHZ1 and KHZ2 were ubiquitously expressed throughout the tissues of Arabidopsis. KHZ1 and KHZ2 were localized to the nucleus, and possessed both transactivation activities and RNA-binding abilities. Taken together, we conclude that KHZ1 and KHZ2 have redundant roles in the regulation of flowering and senescence in Arabidopsis.

As one of the most important events during the life cycle of flowering plants, the floral transition is of crucial importance for plant propagation and requires the precise coordination of multiple endogenous and external signals. There have been at least four flowering pathways (i.e. photoperiod, vernalization, gibberellin, and autonomous) identified in Arabidopsis. We previously reported that two Arabidopsis RNA-binding proteins, KHZ1 and KHZ2, redundantly promote flowering. However, the underlying mechanism was unclear. Here, we found that the double mutant khz1 khz2 flowered late under both long-day and short-day conditions, but responded to vernalization and gibberellin treatments. The late-flowering phenotype was almost completely rescued by mutating FLOWERING LOCUS C (FLC) and fully rescued by overexpressing FLOWERING LOCUS T (FT). Additional experiments demonstrated that the KHZs could form homodimers or interact to form heterodimers, localized to nuclear dots, and repressed the splicing efficiency of FLC pre-mRNA. Together, these data indicate that the KHZs could promote flowering via the autonomous pathway by repressing the splicing efficiency of FLC pre-mRNA.

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Tudor staphylococcal nucleases (TSNs) are evolutionarily conserved RNA binding proteins, which include redundant TSN1 and TSN2 in Arabidopsis. It has been showed TSNs are the components of stress granules (SGs) and regulate plant growth under salt stress. In this study, we find a binding protein of TSN1, RH31, which is a DEAD-box RNA helicase (RH). Subcellular localization studies show that RH31 is mainly located in the nucleus, but under salinity, it translocates to the cytoplasm where it accumulates in cytoplasmic granules. After cycloheximide (CHX) treatment which can block the formation of SGs by interfering with mRNP homeostasis, these cytoplasmic granules disappeared. More importantly, RH31 co-localizes with SGs marker protein RBP47. RH31 deletion results in salt-hypersensitive phenotype, while RH31 overexpression causes more resistant to salt stress. In summary, we demonstrate that RH31, the TSN1 binding protein, is a component of plant SGs and participates in regulation of salt-stress tolerance in Arabidopsis.