Identification and Phylogenetic Analysis of Rice FLZs
To identify candidate FLZ family proteins in rice, the Pfam term DUF581 was used as a query to search the Rice Genome Annotation Project database (RGAP, http://rice.plantbiology.msu.edu/). In total, 29 independent rice OsFLZs were identified. This result was confirmed using the PLAZA v 4.5 database. The 29 FLZs are named OsFLZ1 to OsFLZ29 based on their distribution and relative linear order on rice chromosomes (Additional file 1: Table S1, Additional file 2: Fig. S1). Detail information about each OsFLZ, including gene name, locus, chromosome location (start site and end site), nucleotide length, predicted protein molecular weight, isoelectric point, and protein subcellular localization is given in Additional file 1: Table S1. The coding region of the OsFLZ genes varied from 309 (OsFLZ21) to 1167 (OsFLZ1) base pairs (bps), encoding 103 to 389 amino acids, with molecular weights from 10.76 to 39.42 kilo Dalton (kDa) and isoelectric points from 4.07 to 11.56 (Additional file 1: Table S1). Subcellular localization was predicted using WoLF pSORT (http://www.genscript.com/psort/wolf_psort.html), which showed that OsFLZs proteins were potentially localized in the nucleus, cytoplasm, chloroplast, mitochondria, extracellular and endoplasmic reticulum (Additional file 1: Table S1).
To explore the phylogenetic relationship among FLZ proteins, full-length FLZ protein sequences from rice and Arabidopsis were used to construct an unrooted phylogenetic tree via Neighbor Joining method (Fig. 1a). These proteins grouped in four clades based on their sequence identity. Clade III is the largest group with 11 OsFLZs, and each of the other three clades contains 6 members (Fig. 1a). A multiple sequence alignment of the core FLZ domain, representing around 40 amino acids from FLZ proteins from rice and Arabidopsis, is illustrated in Fig. 1b. Except for OsFLZ19 and OsFLZ23, all other 27 OsFLZ proteins share highly conserved sequence identity (CX2CX17–19FCSX2C), while OsFLZ19 and OsFLZ23 share a single amino acid difference (Fig. 1b). Furthermore, many other residues are equally conserved in the FLZ domain region between rice and Arabidopsis.
The Expression Pattern of OsFLZ Genes in Different Tissues
To understand the potential function of OsFLZ genes in regulating rice growth and development, the spatial expression patterns of 29 OsFLZ genes were analyzed. We downloaded the RNA-Seq data for 11 different tissues/organs of japonica rice cv. Nipponbare from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) to generate a heatmap using Cluster 3 software. As shown in Fig. 2, most OsFLZ genes were ubiquitously but differentially expressed in various tissues and OsFLZ1, OsFLZ2, OsFLZ3, OsFLZ4, OsFLZ9, OsFLZ11, OsFLZ12, OsFLZ14, OsFLZ16, OsFLZ22 and OsFLZ27 were constitutively expressed in all 11 tested samples. The highest expression levels of OsFLZ6 and OsFLZ13 were observed in anther, while OsFLZ5 and OsFLZ10 exhibited the highest expression in post-emergence inflorescence and shoots, respectively. OsFLZ29 was only found to be expressed in the embryo 25 days after pollination (Embryo-25 DAP) at a very low level, and no expression was detected for OsFLZ7 in any of the tissues examined. The diverse expression patterns of OsFLZ genes indicated that they might exert different functions in rice growth and development.
Subcellular Localization of OsFLZs-GFP Fusions
Subcellular localization of OsFLZs predicted by WoLF PSORT suggested that they were localized in diverse subcellular compartments (Additional file 1: Table S1), indicating multifunctional roles in different cellular processes. To confirm the subcellular localization of a subset of OsFLZ proteins, eight representative OsFLZs from four different clades, OsFLZ20 and OsFLZ27 from clade I, OsFLZ10 and OsFLZ2 from clade II, OsFLZ11 and OsFLZ6 from clade III, OsFLZ5 and OsFLZ18 from clade IV, were chosen to fuse in-frame with green fluorescence protein (GFP) driven by a maize ubiquitin promoter, and the resultant chimeric proteins were transiently expressed in 6-week-old tobacco (Nicotiana benthamiana) leaf epidermal cells. Confocal scanning microscopy revealed that the GFP signals of OsFLZ2, OsFLZ5, OsFLZ6, OsFLZ11, OsFLZ18, OsFLZ20 and OsFLZ27 fusions were found both in the nucleus and the cytoplasm, as was the case of empty GFP protein (Fig. 3a). Interestingly, OsFLZ10-GFP was shown to target the granular spots in the cytoplasm, which partially co-localized with chloroplast (Fig. 3b). The results are, in general, consistent with the bioinformatics prediction, in which they mainly localize in nucleus and cytoplasm (Additional file 1: Table S1, Fig. 3).
The Interactions Between OsFLZs and SnRK1A
The essential role of SnRK1A in rice submergence tolerance has been well established (Lu et al. 2007; Lee et al. 2009; Cho et al. 2012). Previous studies showed that FLZs could interact with the subunits of the SnRK1 kinase complex in Arabidopsis and maize (Arabidopsis Interactome Mapping Consortium 2011; Nietzsche et al. 2014, 2016; Jamsheer et al. 2018a, b; Chen et al. 2021). To see whether OsFLZs can also interact with SnRK1A to regulate rice response to submergence stress, we selected the above eight representative OsFLZ genes to perform a yeast two-hybrid assay (Fig. 4). The full-length OsFLZs were fused to the GAL4 activation domain (OsFLZ-AD), and SnRK1A was fused to the DNA binding domain (SnRK1A-BD) vectors, respectively. Six out of the 8 selected OsFLZs-AD (OsFLZ2, OsFLZ5, OsFLZ6, OsFLZ11, OsFLZ18 and OsFLZ20) strongly interacted with SnRK1A-BD, but not with empty BD vector, as judged by the growth of co-transfected yeast cells on SD–Trp/–Leu/–His/–Ade selective medium plates (Fig. 4a). The other two OsFLZ proteins (OsFLZ10 and OsFLZ27) moderately interacted with SnRK1A, as indicated by the growth of co-transfected yeast cells on SD–Trp/–Leu/–His medium containing 1 mM 3-AT, which was comparable to that of the positive control (Fig. 4b). These results suggest that OsFLZs universally interact with SnRK1A, and these interactions might affect rice tolerance to submergence stress.
The Response of OsFLZ Genes Toward Submergence Stress
The above experiments confirmed the strong interactions between OsFLZs and SnRK1A and implied that OsFLZs might function in submergence stress in rice. To confirm this hypothesis, we firstly downloaded and analyzed the transcriptomic data which was derived from Nipponbare with respect to aerobic and anoxic germination (Lasanthi-Kudahettige et al. 2007). We found that 10 OsFLZ genes were significantly down-regulated, but 5 OsFLZ genes were significantly up-regulated, by at least twofold, under anoxia conditions (Fig. 5a), in which OsFLZ18 exhibited the most significant up-regulation. To confirm the responses of OsFLZ genes towards submergence stress, quantitative real-time PCR (qRT-PCR) was used to monitor the transcript abundance of the eight representative OsFLZ and SnRK1A genes in aerobic or hypoxic coleoptiles of Nipponbare. The tested seeds were divided into two groups. One group was submerged in 10 cm water and the other group was kept in air. The coleoptiles from the two groups were collected for total RNA extraction on the 2nd, 4th, 6th, 8th day after treatment, respectively, and qRT-PCR assays were conducted with these samples. The results confirmed that the eight selected OsFLZ genes were all submergence-responsive (Fig. 5b). It is worth noting that the transcription of OsFLZ18 is very drastically up-regulated, with approximately 400-fold increase on the 2nd day and 800-fold increase on the 8th day, respectively (Fig. 5b). Compared to OsFLZ genes, the expression of SnRK1A was slightly repressed in the hypoxic coleoptile (Fig. 5b). These results suggest that both OsFLZ genes and SnRK1A are transcriptionally regulated by submergence treatment.
Functional Confirmation of OsFLZ18 in Regulating Germination and Early Seedling Growth Under Submergence Stress
OsFLZ18 is the most strongly responsive OsFLZ following submergence stress in this study (Fig. 5b). As a case study, we have confirmed its interaction with SnRK1A and function on submergence stress in rice. As shown in Fig. 6a, His-SnRK1A could bind to GST-OsFLZ18, but not bind to the free GST, indicating that SnRK1A physically interacted with OsFLZ18. This interaction was further confirmed by bimolecular fluorescence complementation (BiFC) assay in tobacco (Fig. 6b). When SnRK1A-nYFP was co-infiltrated with OsFLZ18-cYFP into tobacco leaves, strong YFP signals were observed in the transformed cells. However, no fluorescence was detected in negative controls in which SnRK1A-nYFP was coexpressed with cYFP or OsFLZ18-cYFP was co-expressed with nYFP (Fig. 6b). To assess the potential role of the OsFLZ18-SnRK1A interaction, we employed the widely used dual-luciferase assay to evaluate the effects of OsFLZ18 on the signaling output of SnRK1A, by measuring the transcriptional regulatory activity of SnRK1A to its induced target gene αAmy3 (Lu et al. 2007). In line with the previous report (Lu et al. 2007), SnRK1A could significantly activate the expression of αAmy3 (Fig. 6d). Interestingly, addition of OsFLZ18 could effectively antagonizes the SnRK1A-dependent transcriptional activation of αAmy3 (Fig. 6d), indicating that OsFLZ18 might function as a repressor of SnRK1 signaling.
To further confirm the function of OsFLZ18 in rice submergence stress, we generated OsFLZ18 overexpression (OE) transgenic rice plants in Nipponbare background. In total, 25 independent OsFLZ18-GFP OE lines were obtained, and three homozygous T3 lines with high but different gene and protein expression levels were chosen for further phenotypic evaluation (Fig. 7a, b). We also checked the expression levels of SnRK1A and αAmy3 in Nipponbare and OsFLZ18-GFP OE lines by qRT-PCR assay. The results showed that the expression of SnRK1A had no obvious alteration among Nipponbare and OE lines, but the expression level of αAmy3 was significantly decreased in OE lines (Fig. 7c). To investigate whether OsFLZ18 affects rice growth and development, wild type (WT) and three OsFLZ18-GFP OE transgenic seeds were respectively germinated in air or submergence conditions and their phenotypes were compared after growing for 7 days (Fig. 7d). In air, the growth of OsFLZ18-GFP OE plants were retarded, with significantly shorter shoots than the wild type plants (Fig. 7d, e). Under the submergence condition, all genotypes failed to generate roots, but the coleoptiles emerged and more elongated in WT than those in OsFLZ18-GFP OE lines (Fig. 7d–f). In addition, the shoot lengths in air and coleoptile lengths in submergence were negatively correlated with the gene or protein expression levels. These results together suggest that OsFLZ18 negatively regulate rice growth in both air and submergence conditions.