Vascular bundles of plants, composed of xylem and phloem tissues, connect all parts of the plant body from leaf to root through their conductive function, providing water and nutrients transport, mechanical support, and overall structural strength (1). Auxin, which is important for vascular establishment and maintenance, is mainly synthesized in the plant cells of actively growing tissues, such as developing leaves and roots, and transported to other tissues to participate in organ primordia formation, apical meristem maintenance, and vascular tissue differentiation through the process of polar auxin transport (2-4).
Auxin flow distribution and gradient establishment are regulated by the specific cellular auxin transport proteins (5). PIN proteins are known to determine the direction and distribution of auxin movement (5, 6). In Arabidopsis, five PINs are predominantly localized in the plasma membrane, and determine the direction of intracellular auxin flux to maintain auxin gradient distribution (7). The Arabidopsis pin mutants show growth defects in shoot and stems, root, inflorescence, and embryo (3, 8). The rice genome contains twelve PIN genes (9). The ospin mutants are reported to be involved in root development, plant height, tiller and panicle branch angle, panicle formation, yield, and tiller growth (10, 11). Several mutants have been reported to affect rice vascular development and root formation by regulating the expression of OsPIN genes. For example, the mutation of NARROW LEAF1 (NAL1) caused defective cell division by affecting polar auxin transport OsPINs, and showed reduction in veins and adventitious root formation (12, 13). The NARROW LEAF2/3 (NAL2/3) plays a key regulatory role in lateral axis outgrowth, vascular development, and root formation, through the regulation of OsPIN genes (14-16).
Several studies have reported the key role of chromodomain helicase DNA-binding (CHD) family proteins as transcriptional regulators in the regulation of developmental processes and stress response (17, 18). In Arabidopsis, PICKLE belongs to typical CHD protein, while the pkl mutant shows pleiotropic defects, such as semi-dwarfism and reduced root meristem activity (19, 20). PICKLE positively regulates the expression of auxin-related genes IAA19 and IAA29 by repressing H3K27me3 deposition (21). In rice, RFS/CHR729 is a member of the CHD family proteins, and is one of the close homologs of PICKLE (22). RFS/CHR729 protein interacts with the promoter regions of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3, while chr729 mutant exhibits pleiotropic phenotypes, such as narrow and rolled leaves, reduced stem elongation, and decreased chlorophyll contents (17, 22). Moreover, all other allelic mutants of RFS/CHR729 display clear defects in vascular development (22-24), suggesting that RFS may modulate specific gene expression related to vascular development by activating or repressing epigenetic marks on histones. However, the detailed molecular mechanisms of the CHD family protein RFS in plant vascular development and root formation remain unclear. To clarify the underlying mechanism of RFS in vascular development and root formation, we used RNA-seq, RT-qPCR, and ChIP assays to analyze the expression and histone methylation levels of auxin-related genes in rfs mutant.
A previous study found that rice rfs mutant has reduced leaf vascular bundles and the number of roots throughout the life span (17). To better understand whether vascular defects occur throughout the tissue, we performed the histological analysis of shoot apical meristem (M) and leaf primordia in 3-day-old seedlings, using paraffin section. The shoot apical meristem and leaf primordia (P1 to P3) in rfs mutant were similar in size to those of WT, whereas the number of provasculatures in rfs mutant was lower than in WT (Fig. 1A-D). We examined the number of large vascular bundles (LVB) in the leaf sheath and basal region of 10-week-old rfs mutant, and found the number of LVB in rfs mutant was less than that in WT (Fig. 1E-H). Additionally, the phloem (P) and xylem (X) sizes in rfs leaf sheath were smaller than those in WT (Fig. 1J, L). Notably, the LVB of stem in rfs mutant showed impaired differentiation, with an unclear distinction between phloem and xylem (Fig. 1I, K). Furthermore, the leaf sheath of rfs mutant exhibited fewer AE or no SVB (Fig. 1E-H). As we previously observed fewer adventitious roots in rfs mutant (17), we investigated root growth initiation in both 3-day-old seedlings and 10-week-old rfs mutants. The results indicated a 63% reduction in the number of adventitious root primordia in rfs mutant (Fig. 1M-P). In addition, rfs mutant displayed shorter root cap and meristematic zone (Supplementary Fig. 1). These results suggest that RFS is necessary for maintaining vascular and root development from the beginning of plant growth.
Given that RFS remodels chromatin structure by the methylation or acetylation of histone proteins and thus regulates gene transcription (22), the mutation of RFS may affect the rice transcriptome, including vascular development-related genes. To obtain deeper insights into the function of RFS, the transcriptomic profiles in WT and rfs were characterized. A total of 2,284 DEGs were identified between WT and rfs, including 1,519 up-regulated and 765 down-regulated DEGs in rfs mutant (Fig. 2A, Supplementary Table 1).
Subsequently, GO was used to classify the functions of DEGs. The results implied that the DEGs annotated with GO terms were mainly related to “nucleolus”, “preribosome”, “small-subunit processome”, and “protein refolding” (Supplementary Fig. 2A).
To uncover potential signaling pathways that regulate the impaired vascular development phenotypes of rfs mutant, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. KEGG analysis for DEGs between WT and rfs mutant revealed that the phenylpropanoid biosynthesis, phenylalanine metabolism, and plant hormone signal transduction pathways significantly correlated with the rfs phenotype (Supplementary Fig. 2B). In particular, both phenylpropanoid and phenylalanine pathways produce lignin, which is a composite for xylem in vascular tissue (25). Interestingly, three DEGs in “Phenylpropanoid biosynthesis (Os02t0626100-02, Os02t0626400-01/03)” and one DEG in “Phenylalanine metabolism (Os05t0137000-01)” were down-regulated (Supplementary Table 1). The down-regulated DEGs in “Plant hormone signal transduction” were Os06t0232300-01 and Os08t0529000-01 (Supplementary Table 1), encoding auxin transport proteins, which are critical genes for vascular tissue development in rice (10).
Narrow leaf phenotype is generally associated with auxin signal (12-14, 26). NAL2/3 and NAL1 could affect vascular development through auxin transport genes, OsPINs (12, 13). In addition, a previous study reported that allelic mutants of rfs showed both altered OsPIN and reduced NAL1 expressions (24). First, we investigated the DEGs in auxin-related genes. Indeed, many auxin-related genes were altered in rfs mutant (Fig. 2B). Notably, the level of OsPIN1a and OsPIN5b were downregulated in rfs mutant (Fig. 2B). The RT-qPCR results were consistent with the RNA-seq results for the levels of some auxin-related genes (Fig. 2C).
The mutation of NAL1 and NAL2/3 has been reported to affect the expression of OsPIN family genes (13, 14, 16). Therefore, we examined the contribution of other OsPINs, NAL1, NAL2/3, and NAL7 genes in seedling plants. Several OsPIN genes showed significantly altered expression levels in rfs mutant, compared to WT plants (Fig. 2D). Although OsPIN1b, OsPIN1c, OsPIN2, and OsPIN9 were reduced in rfs mutant, a result similar to that previously reported in nal1 mutant (13), OsPIN5a expression was up-regulated. No statistical difference in the level of OsPIN10 was observed in rfs mutant plants. Consistent with the OsPIN1s transcript levels, OsPIN1 protein level was reduced in rfs (Fig. 2E). Furthermore, NAL1 expression was reduced in rfs mutant, whereas there was no difference in NAL2/3 and NAL7 expression (Fig. 2D). These results suggest that the rfs phenotype is due to the perturbed expression of auxin-related genes, especially NAL1 and OsPIN family genes, in the shoot basal region, where the shoot and root tissue primordia initiate.
Previous studies have shown that both nal1 and OsPIN1-RNAi mutants are defective in adventitious root development (10, 13). Moreover, overexpression of OsAGAP resulted in a decrease in both the length and number of adventitious roots (27). Based on the findings and the observed changes in the expression levels of OsPINs, NAL1, and OsAGAP in rfs mutant (Fig. 2), we further investigated the root phenotype. As expected, rfs showed fewer and shorter adventitious roots, compared to WT (Fig. 3A, C, D).
PIN family genes have been implicated in polar auxin transport in plant cells (28). Specifically, auxin treatment restored the defects in adventitious root formation in OsPIN1-RNAi and nal1 mutants (10, 13). Thus, we examined adventitious root development in response to exogenous auxin (IAA) in rfs seedlings. While the adventitious root length of rfs seedlings was rescued by exogenous application of IAA, the number of adventitious roots of rfs seedlings was partially restored (Fig. 3B-D).
Auxin has been reported to induce OsPINs expression (29). To confirm whether the restored root phenotype was due to the induction of OsPINs expression, we investigated the expression of OsPINs after exogenous auxin and auxin inhibitor treatments. The expression of OsPIN1b and OsPIN1c was increased by IAA treatment in rfs mutant, consistent with the morphological responses in roots (Fig. 3F, G). However, after exogenous IAA treatment, OsPIN1a and NAL1 were not increased in rfs mutant (Fig. 3E, H). As expected, following treatment with exogenous auxin inhibitor NPA, the expression of all genes was repressed in both WT and rfs mutant (Fig. 3I-L). These results suggest that altered expression of OsPINs and NAL1 genes affects the appropriate distribution of endogenous auxin in rfs, which in turn may affect adventitious root development.
Previous studies have shown that rfs mutant has a decreased level of overall histone methylation, in particular, a global reduction at the activating mark H3K4me3 (17, 22). Interestingly, unlike OsPIN1b and OsPIN1c, there was no increase in the expression of OsPIN1a and NAL1 in rfs mutant after auxin application (Fig. 3E, H). Thus, we performed a chromatin immunoprecipitation (ChIP) assay to investigate whether RFS affects the levels of H3K4me3 on the auxin-related genes, such as NAL1 and OsPIN1a. The levels of H3K4me3 were found to be reduced in the promoter region of NAL1 and OsPIN1a in rfs mutant, compared with WT (Fig. 4A-D). These findings suggest that RFS affects the modification of histone proteins that bind directly to auxin-related genes, which are essential for maintaining vascular and adventitious root development.
Given the previous studies on the rice CHD3/Mi-2 chromatin remodeling factor CHR729/RFS (17, 22-24, 30, 31), RFS is necessary to regulate gene expression related to auxin at the histone level, and contributes to the regulation of vascular development and root development in plants. In this study, we identified rfs mutant with defects in vascular development in the regions of leaf and stem, and root formation (Fig. 1, Supplementary Fig. 1). Mutation of the PKL, an Arabidopsis ortholog of RFS, affects the formation of root meristem tissue (32), which is partially similar to the rfs mutant. In addition, pkl mutants exhibit short height and small leaves, compared to WT (33). Furthermore, it was reported that PKL appears to control tissue specification through changes in the histone modifications of auxin-related gene (34). Interestingly, the transcriptome analysis revealed that rfs mutation affects the expression of auxin-related genes similar to the pkl (Fig. 2). This suggests that CHD family proteins may have similar functions in plant development, specifically in vascular development and root formation through the auxin signaling pathway.
Notably, rfs mutant exhibited the reduced expression of ARFs (Fig. 2B, C). Previous studies showed that ARF3, ARF6, and ARF8 are involved in lignin biosynthesis in various biological processes in plants (35, 36). Our transcriptome analysis showed that the phenylalanine pathway, which is involved in lignin biosynthesis, was significantly affected in rfs mutant (Supplementary Fig. 2B). Lignin is a key component for vascular plants, and biopolymer lignin is deposited in the cell walls of vascular cells (37). Moreover, histological analysis revealed that some of the vascular bundles were absent or defective in rfs mutant (Fig. 1E-L). These data suggest that RFS may affect lignin synthesis, which participates in vascular development through the auxin signaling pathway.
OsPIN1 distributes proper concentration of auxin for the development of vascular tissues and root emergence (10, 38). In particular, it was reported that four OsPIN1 showed functional redundancy (38). Among the single mutants, ospin1b had decreased adventitious root formation, panicle length, and grain number, and increased grain yield and tiller number, whereas the other single mutants had no critical phenotypes (10, 29). Only pin1apin1b or pin1cpin1d double mutants exhibited critical phenotypes, such as reduced plant height, root number and root length, and increased tiller number (38). It was reported that the mutation of NAL1 preferentially affects the expression of overall OsPIN genes, resulting in defects in vascular bundle development and root formation (13). Consistent with these reports, most OsPINs were significantly down-regulated in rfs mutant (Fig. 2). Thus, these data indicate that RFS may regulate auxin homeostasis in rice to control vascular development and root formation through the regulation of OsPINs. In addition, rfs mutant showed defects in yield traits similar to nal1 (Supplementary Table 2) (17, 39). Interestingly, unlike rfs mutant, ospin1b CRISPR mutant exhibited increased grain yield and tiller number, compared to WT (29). This is a different phenotype from rfs mutant, suggesting that while RFS directly regulates OsPIN1a, it may also affect most of OsPINs expression, primarily by regulating NAL1. A recent study reported that the NAL1-OsTPR2 module directly regulates PIN1b at the histone level (39). Therefore, while RFS may be involved in this NAL1 complex module as a coactivator or corepressor, the details require further research.
RT-qPCR and ChIP analysis confirmed that RFS directly activates the transcription of auxin-related genes, OsPIN1a and NAL1, which regulate vascular development and root formation (Figs. 3 and 4), indicating that the RFS may directly regulate vascular development and root formation through both OsPIN1a and NAL1. Taken together, RFS may be a higher hierarchical regulator of the auxin signaling pathway cascades in plant vascular development and root formation.
Materials and methods are available in supplementary information.
This research was supported by the Basic Science Research Program through the NRF of Korea grant funded by the Korean Government (NRF-2010-355-F00003), the Brain Pool program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (2022H1D3A2A01096185), and the Basic Science Research Program through the NRF of Korea grant funded by the Korean Government (RS-2023-00247376).
The authors have no conflicting interests.