A network comprising ELONGATED HYPOCOTYL 5, microRNA397b, and auxin-associated factors regulates root hair growth in Arabidopsis

Abstract

ELONGATED HYPOCOTYL 5 (HY5) is a major light-associated transcription factor involved in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of HY5 is very well defined in regulating primary root growth and lateral root formation; however, information regarding its role in root hair development is still lacking, and little is known about the genetic pathways regulating this process. In this study, we investigated the role of HY5 and its associated components in root hair development. Detailed analysis of root hair phenotype in wild-type and light signaling mutants under light and dark conditions revealed the importance of light-dependent HY5-mediated root hair initiation. Altered auxin levels in the root apex of the hy5 mutant and interaction of HY5 with promoters of root hair developmental genes were responsible for differential expression of root hair developmental genes and phenotype in the hy5 mutant. The partial complementation of root hair in the hy5 mutant after external supplementation of auxin and regaining of root hair in PIN-FORMED 2 and PIN-FORMED 2 mutants after grafting suggested that the auxin-mediated root hair development pathway requires HY5. Furthermore, miR397b overexpression (miR397bOX) and CRISPR/Cas9-based mutants (miR397b^CR) indicated miR397b targets genes encoding reduced residual arabinose (RRA1/RRA2), which in turn regulate root hair growth. The regulation of the miR397b-(RRA1/RRA2) module by HY5 demonstrated its indirect role by targeting root hair cell wall genes. Together, this study demonstrated that HY5 controls root hair development by integrating auxin signaling and other miRNA-mediated pathways.

Introduction

Light is one of the crucial environmental signals regulating plant growth and development (Galvão and Fankhauser 2015). Light affects several responses in plants, including germination, seedling de-etiolation, stem elongation, phototropism, stomata and chloroplast movement, shade avoidance, circadian rhythms, and flowering time (Deng and Quail 1999). Light-facilitated seedling growth is termed photomorphogenesis or de-etiolation (Quail 2002; Sullivan and Deng 2003). The growth of seedlings in the dark is recognized as skotomorphogenesis (Josse and Halliday 2008). Plants perceive the light of diverse wavelengths through a collection of proteins termed photoreceptors (Shinomura et al. 1994; Cashmore et al. 1999; Casal 2000; Kliebenstein et al. 2002; Li et al. 2011). These photoreceptors transmit light signals further through a signaling cascade to regulate the expression of several genes, leading to physiological changes. Among numerous transcription factors that act downstream to photoreceptors, a bZIP transcription factor, ELONGATED HYPOCOTYL 5 (HY5), is well known for its role in regulating light-mediated growth and response (Gangappa and Botto 2016; Kreiss et al. 2023; Singh et al. 2024). CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiquitin ligase, acts as a negative regulator of light signaling by promoting the proteasome-mediated cleavage of several light-mediated positive transcription factors that include HY5 (Deng et al. 1992; Osterlund et al. 2000; Hoecker 2017; Kim et al. 2017). The COP1–HY5 unit is essential to the proper functioning of the light system in the photomorphogenic process (Saijo et al. 2003; Xu 2020).

Root hairs are elongated tubular-shaped extensions on root epidermal cells. They vastly upsurge the root's external area and efficiently increase the root width, assisting plants in nutrient acquisition (Hofer 1991). Cell walls play an important role in root hair cell development because it contains majorly hydroxyproline-rich glycoproteins. This includes extensins (EXTs), proline-rich proteins, and arabinogalactan proteins. In root hairs, first EXTs are modified by the proline hydroxylases followed by arabinosyltransferases, XEG113/RRA1-3. The posttranslationally modified EXTs are secreted into the cell wall where it cross-links with Tyr residues, and this is a very crucial step for the assembly of the cell wall for root hair formation (Kieliszewski and Lamport 1994; Velasquez et al. 2011).

The phytohormone auxin is a well-characterized hormone, and it positively regulates root hair growth and development (Paque and Weijers 2016; Weijers and Wagner 2016; Gaddam et al. 2021). Auxin promotes root hair growth by promoting the transcription of auxin response factors (ARFs), and they act as activators or repressors of the transcription of genes (Choi et al. 2018). Auxin can restore root hair even in rhd6 mutant (Masucci and Schiefelbein 1996) that lacks the function of central root hair initiating basic helix–loop–helix (bHLH) transcription factor by activating another bHLH transcription factor ROOT HAIR DEFECTIVE 6-LIKE4 (RSL4) (Yi et al. 2010). ARFs(ARF5) activates RSL4 by directly binding to its promoter sequence (Mangano et al. 2017).

HY5 negatively regulates auxin signaling by promoting the negative regulators of auxin signaling (Cluis et al. 2004). HY5 controls the accumulation of auxin in the root of Arabidopsis (Arabidopsis thaliana) by controlling the intracellular distribution of auxin transporter PINFORMED2 (PIN-2) (Laxmi et al. 2008). In addition, hy5 mutants show increased local auxin accumulation at lateral root primordia (Zhang et al. 2019). Previous studies have characterized the role of HY5 in primary root growth and lateral root development, but its function in root hair development is still unclear. In this study, we demonstrated the importance of HY5 in root hair development. Firstly, HY5 regulates the expression of miR397b, which can target root hair cell wall-related genes in A. thaliana. Secondly, HY5 is necessary for auxin-mediated root hair growth and development. We concluded that the loss of function of HY5 leads to differential expression of auxin-responsive root hair developmental genes. Together, these findings suggest that HY5 integrates with auxin signaling in the regulation of root hair growth.

Results

RRA1/RRA2-mediated root hair elongation is regulated by miR397b

In Arabidopsis, RRA1/RRA2 are known to regulate root hair elongation (Velasquez et al. 2011). It was proposed that miR397b might have a role in root hair elongation by targeting RRA1/RRA2 (Huang et al. 2021). To define the role of miR397b in root hair elongation, we developed Cas9-free knock-down mutants of miR397b using CRISPR/Cas9 technique (Supplementary Fig. S1). The relative expression analysis of miR397b in edited lines suggested substantial downregulation of mature miR397b that leads to upregulation of predicted target RRA1/RRA2 genes (Fig. 1, A and B). To validate that miR397b targets RRA1/RRA2, we have generated the overexpression (miR397bOX) lines of the precursor sequence of miR397b in Arabidopsis. The expression analysis of miR397bOX lines suggested enhanced expression of miR397b, and a significant reduction in the level of RRA1/RRA2 transcripts in all the selected transgenic lines (Fig. 1, C and D). To further confirm that both RRA1/RRA2 were targets of miR397b, we performed a 5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) assay. Sequencing analysis of the PCR products obtained from RLM-RACE confirmed that the miR397b targets both RRA1/RRA2 mRNAs, a few nucleotides away from the cleavage site (Fig. 1E). Similar to rra1 and rra2 mutants, miR397bOX seedlings also exhibit shorter root hairs compared to wild-type (WT), and miR397b^CR mutants showed enhanced root hair length compared to WT and miR397bOX seedlings (Fig. 1, F and G). All together these results clearly suggested that miR397b targets RRA1/RRA2 and regulates root hair growth.

HY5 controls miR397b-RRA1/RRA2 module in incoherent feed-forward loop manner

Recently, an indirect role of HY5 was suggested in root hair growth by modulating the levels of cell wall-related gene galactosyl transferase (Gaddam et al. 2021). This led us to think that HY5 might have a role in the regulation of the miR397b-RRA1/RRA2 module to control root hair growth. To validate this hypothesis, expression analysis of mature miR397b and its target genes, RRA1 and RRA2, was carried out in roots of 5-d-old light- and dark-grown WT and hy5-215, hy5 (hereafter) seedlings. The relative expression analysis suggested that transcript levels of miR397b were significantly downregulated in hy5 mutant under both light and dark conditions (Fig. 2A). Interestingly, in the hy5 mutant root, the same pattern was observed in which the expression of RRA1 and RRA2 significantly decreased in the hy5 mutant under light conditions and no differential regulation was observed in both WT and hy5 mutant under dark conditions (Fig. 2B). This scenario illustrates a feed-forward loop motif, exemplifying how a master transcription factor can control the expression of both miRNA and its target genes. This regulation can occur through coherent pathways, where the transcription factor activates the miRNA while inhibiting its targets, or through incoherent pathways, where the transcription factor activates both the miRNA and its target genes (Osella et al. 2011). The above results suggested that HY5 regulates the miR397b-RRA1/RRA2 module in an incoherent feed-forward loop manner (Fig. 2C).

To understand the functional role of miR397b in Arabidopsis, we carried out a detailed analysis of its promoter region. The in silico analysis of the promoter led to the identification of a light-responsive element (LRE), G-box (CACGTG), upstream to the transcription start site (TSS) (Fig. 2D). To validate the regulation of miR397b gene expression by HY5 at the molecular level, the interaction of miR397b promoter with HY5 was studied through in vitro electrophoretic mobility shift assay (EMSA). The HY5 protein bound to T/G-Box-containing probe but not to its mutated probe (T/G-Box-mut), as observed through EMSA (Fig. 2E). To further confirm the binding of HY5 to the promoter of miR397b, a chromatin immunoprecipitation (ChIP)-qPCR assay was performed. The ChIP-qPCR results suggested that HY5 directly binds to the promoter regions containing the G-box (P1). The qPCR analysis showed the P1 region (containing G-box) was significantly enriched in the ChIP samples from HY5OE/hy5 seedlings than the P2, P3, and P4 portions (Fig. 2F). This analysis demonstrates that HY5 directly binds to the G-box of the miR397b promoter. Together, all these results suggest that HY5 directly binds to the LREs present in the promoter of miR397b.

HY5 has a role in root hair development

Previous studies have reported that the hy5 mutant was unable to develop root hair after treatment with low Pi (Yeh et al. 2020). This finding made us to hypothesize that HY5 might directly regulate root hair development. To prove our hypothesis, we analyzed the root hair phenotypes in 5-d-old light- and dark-grown WT, hy5, hy5/HY5OX, and cop1-4 seedlings. We observed a remarkable reduction in root hair number and length in the hy5 mutant compared to WT, hy5/HY5OX, cop1-4 (Fig. 3, A to C). The lack of root hair in the hy5 mutant indicated its role in root hair development. As anticipated, our investigation revealed a positive association between light exposure and the development of root hairs. Analysis of root morphology indicated a notable decrease in the number of root hairs in WT, hy5/HY5OX, and cop1-4 seedlings grown in darkness compared to those grown under light conditions. However, intriguingly, the hy5 mutant exhibited no significant disparity in root hair count between light and dark conditions (Fig. 3, A and B). Moreover, the length of root hairs exhibited a significant reduction in hy5, WT, and cop1-4 under dark conditions compared to light, while hy5/HY5OX showed no significant variance in root hair length (Fig. 3C). These results suggest that light mediates root hair development through the HY5 transcription factor in Arabidopsis. To provide additional evidence for our proposed hypothesis, a hypocotyl grafting between root stocks of hy5 with hy5 mutant and WT scions was performed. Two weeks after the grafting, root hair phenotype was analyzed. The root hair phenotype suggested a significant root hair complementation in the hy5 root stock when it was grafted with the scion of WT and not the hy5 (Fig. 3D). These findings indicated an essential role of HY5 in root hair development.

Altered levels of auxin lead to root hair development defects in hy5

Proper auxin distribution and abundance can maintain the priming and outgrowth of root hair cells (Josse and Halliday 2008; Grierson 2009; Grierson et al. 2014; Leyser 2018). Previous studies have suggested that (Indole-3-acetic acid) IAA levels are modulated in hy5 mutants, and also, auxin has a role in root hair initiation. To study whether the aberrant root hair phenotype of the hy5 mutant is a result of modulation of the auxin pathway, we quantified the free IAA content in 5-d-old dark and light-grown seedlings of hy5, hy5/HY5OX, WT, and cop1-4 through LC–MS/MS analysis (Fig. 4A, Supplementary Fig. S2). The analysis suggested that IAA content is significantly low in dark-grown seedlings compared to seedlings that were grown in the light. The hy5 mutant exhibited significantly low levels of IAA compared to hy5/HY5OX, WT, cop1-4 under light conditions. This result was further validated by relative expression analysis of auxin-associated genes in 5-d-old light-grown roots and seedlings of WT, hy5, hy5/HY5OX, cop1-4. Interestingly, transcript levels of auxin signaling components, auxin transporters, and also biosynthesis-related genes were downregulated in the hy5 mutant root and seedlings (Fig. 4B, Supplementary Fig. S3). The change in the expression of auxin pathway genes in root might be the reason for the difference observed in the whole seedlings (Supplementary Fig. S3).

To further validate the role of HY5 in the regulation of the auxin biosynthesis pathway, the grafting experiment was performed in hy5, hy5/HY5OX, cop1-4, and WT with auxin marker DR5 promoter lines. The scion of WT, hy5, hy5/HY5OX, and cop1-4 was grafted with a stock of DR5 promoter lines (Fig. 4C). The histochemical GUS staining showed that the GUS expression was almost negligible in DR5 reporter–root apex when hy5 was used as the scion. In contrast, enhanced expression of GUS was observed at the root apex of reporter lines when hy5/HY5OX, WT, and cop1-4 were used as scions (Fig. 4, C and D). The accumulation of auxin at the root apex induces cell division that positively correlates with root hair growth (Mishra et al. 2009; Barrada et al. 2015). These results clearly indicate that the defective root hair development in hy5 is due to lesser auxin accumulation at the root apex.

The altered auxin pathway modulates expression of root hair developmental genes

Studies have shown that auxin induces the expression of root hair developmental genes such as ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4), ROOT HAIR DEFECTIVE SIX-LIKE2 (RSL2), and EXPA7 (EXPANSIN A7) (Mangano et al. 2017). To further know whether altered auxin accumulation in hy5 mutant leads to differential expression of different root hair developmental genes, we analyzed their expression in 5-d-old light-grown WT, hy5, hy5/HY5OX, and cop1-4 seedlings. The relative expression analysis suggested a significant upregulation of all the root hair development-related genes in WT, hy5/HY5OX, cop1-4 compared to the hy5 mutant (Supplementary Fig. S4). To specifically know the expression pattern of root hair developmental genes in Arabidopsis root, we analyzed the expression in roots of 5-d-old light as well as dark-grown seedlings of WT, hy5, hy5/HY5OX, cop1-4. The expression analysis suggested a significant up-regulation of RSL2, RSL4, and EXPA7 in roots of light-grown seedlings of WT, hy5/HY5OX, cop1-4 compared to hy5 mutant. In contrast, roots of dark-grown seedlings failed to induce the expression of these root hair developmental genes (Fig. 4, A to C). The differential expression of RSL2, RSL4, and EXPA7 in the hy5 mutant root might be due to a low accumulation of IAA in the root. But roots of dark-grown seedlings of WT, hy5, hy5/HY5OX, cop1-4 failed to induce the expression of root hair developmental genes (Fig. 5, A to C). This further suggests that HY5 might have a positive regulation on root hair developmental genes. To validate the regulation of RSL4, RSL2, and EXPA7 genes expression by HY5 at the molecular level, the interaction of HY5 with promoters of these genes was studied through EMSA. The HY5 protein bound to the G-Box-containing probe but not its mutated version (G-Box-mut) as depicted by EMSA (Fig. 5, D to F, Supplementary Fig. S5A–C). To further confirm the binding of HY5 to the promoter of RSL2, RSL4, and EXPA7, ChIP-qPCR was performed. The ChIP-qPCR results indicate that HY5 directly binds to the promoter regions of RSL4, RSL2 containing G-box (P2) and EXPA7 G-box (P3). The qPCR analysis showed that the sections containing the G-box were significantly more enriched in the samples of hy5/HY5OX seedlings than the promoter portion without the G-box (Fig. 5, G to I). These results indicate that HY5 directly binds to the LREs present in the promoter of the RSL4, RSL2, and EXPA7 genes and also support our finding that HY5 directly regulates the transcription of root hair development genes.

External auxin partially rescued root hair density phenotype in hy5 mutant

It is known that an external supply of auxin can induce root hair in Arabidopsis (Masucci and Schiefelbein 1996; Knox et al. 2003; Grieneisen et al. 2007). To study whether the external supply of auxin can induce root hair in hy5 mutant, 4-d-old WT, hy5, hy5/HY5OX, and cop1-4, seedlings were transferred to half-strength media containing different concentrations of IAA. Surprisingly, externally supplied IAA failed to induce root hair density at higher concentrations in hy5 mutant as most of the epidermal region was found to be root hairless. But at low concentrations of IAA, hy5 mutant could partially regain root hair growth. The root hair length and the number increased significantly in WT, hy5/HY5OX, and cop1-4 seedlings compared to the control condition when they were treated with auxin, especially at low concentrations (Fig. 6, Supplementary Fig. S6). To study whether the hy5 mutant is generally resistant to external IAA, we also measured other root parameters like lateral root number. A significant increase in lateral root number in the hy5 mutant compared to the control condition was observed (Supplementary Fig. S7). These data suggest that hy5 in general does not have resistance toward external auxin application. These results demonstrate that auxin-mediated root hair growth initiation requires light-mediated transcription factor HY5 in Arabidopsis.

HY5 acts as an important integral component in auxin-mediated root hair development

Earlier results emphasized that HY5 regulates auxin biosynthesis and is regulatory component in auxin-mediated root hair development. From these two observations, we hypothesized that HY5 might be regulated by auxin. To find the effect of auxin on the expression of the HY5, we performed expression analysis using 5-d-old WT seedlings treated with 0.1N NaOH (control) and IAA (1 µM) at different time points. The expression analysis suggested an enhanced in the transcript levels of HY5 as the time of auxin treatment is increased (Fig. 7A).

To further validate the role of auxin in the regulation of HY5, we analyzed the transcript levels of HY5 in Arabidopsis seedlings that were treated with auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA; 25 µM) as well as in auxin transport mutants PIN-FORMED 2 (pin2), PIN-FORMED 3 (pin3). The expression analysis suggested that the expression of HY5 significantly downregulated in TIBA-treated seedlings as well as in pin2 and pin3 (Fig. 7, B and C). Auxin efflux carrier PIN-FORMED 2 (PIN2) has an important role in root hair development by maintaining proper auxin gradient in root tips (Chen et al. 2022). The observation of root hair phenotype of pin2 and pin3 mutants also suggested that loss of activity of pin2 and pin3 leads to aberrant root hair phenotype in Arabidopsis (Supplementary Fig. S8). To further investigate the role of HY5 in the auxin-mediated root hair development pathway, grafting was performed between the scion of hy5, hy5/HY5OX, cop1-4, and WT with the root of pin2, pin3 mutants. The grafting analysis suggested that root hairs were regained in pin2 and pin3 mutants roots when they were grafted with WT, hy5/HY5OX, and cop1-4 scions, while the development of root hair failed in the roots of pin2, pin3 mutants when they were grafted with hy5 scion (Fig. 7, D and E). These results proved the importance of HY5 in the auxin-mediated root hair development pathway.

Discussion

This study elucidates previously unexplored mechanisms governing root hair development in A. thaliana. Through various experimental approaches, we arrived at the conclusion that HY5 regulates the miR397b-RRA1/RRA2 module, thus exerting control over the development of root hair length. Additionally, our investigation shed light on HY5's involvement in root hair initiation, achieved through the modulation of root hair developmental genes in conjunction with the auxin signaling pathway (Fig. 8). Altogether, our findings identified the significant role of the shoot-to-root mobile transcription factor HY5 in integrating multiple pathways crucial for root hair development.

HY5-miR397b-RRA/RRA2 module regulates root hair length

Extensins (EXTs) are highly O-glycosylated proteins involved in root hair development (Møller et al. 2017; Velasquez et al. 2011). RRA1/RRA2 belong to the GT77 family and are supposed to transfer second arabinose to the extension protein's backbone (Egelund et al. 2007). Arabinosylation of extension is important for proper root hair length and the knock-down of RRA1 and RRA2 in rra1rra2 mutant results in shorter root hair phenotype (Møller et al. 2017). The molecular mechanism that can regulate these arabinosyltransferases is still not known. In this study, we explored the upstream pathways that have the potential to control RRA1/RRA2 to regulate root hair development in Arabidopsis. Previous studies have predicted that both RRA1/RRA2 proteins might be targeted by miR397b (Huang et al. 2021). Relative expression analysis in the miR397b^CR and miR397bOX transgenic lines, followed by 5′RLM-RACE, suggests that miR397b targets both RRA1/RRA2 (Fig. 1, A to E, Supplementary Fig. S1). The root hair length genotypes of WT, miR397bOX, and miR397b^CR exhibited a strong correlation. These results provided additional evidence of miR397b's function in controlling the growth of root hair (Fig. 1, E and F). This study found that miR397b transcript levels decreased in the hy5 mutant, confirming its light sensitivity. The differential expression of miR397b and its target genes RRA1/RRA2 in the mutant suggests that HY5 positively regulates these expressions to maintain proper root hair growth. (Fig. 2, A and B). We further demonstrate that HY5 directly binds to the promoter of miR397b (Fig. 2, D to F).

HY5 regulates root hair abundance by converging with the auxin regulatory pathway and root hair developmental genes

The subsequent inquiry cantered on whether HY5 plays a role in Arabidopsis root hair development. Zhang et al. (2019) previously demonstrated that the hy5 mutant exhibits longer root hairs compared to the WT when subjected to a root covering system. Conversely, another study showcased the absence of root hair initiation in the hy5 mutant under low Pi treatment conditions (Yeh et al. 2020). These contrasting findings could potentially stem from variations in the growth conditions employed in the respective experiments. In our investigation, we observed a notable reduction in root hair growth upon loss of HY5 function in the hy5 mutant. Conversely, the WT, hy5/HY5OX, and cop1-4 lines displayed well-developed root hairs (Fig. 3, A to C). These observations led us to hypothesize that HY5 indeed plays a pivotal role in root hair development. Notably, similar phenotypes observed in dark-grown WT seedlings further suggested the involvement of HY5 in root hair growth. Additionally, the successful rescue of root hair development in hy5 mutant rootstock when grafted with WT scions provided further support for our hypothesis (Fig. 3D). It is well established that auxin acts as a positive regulator of root hair development in Arabidopsis (Paque and Weijers 2016). Alterations in auxin levels and signaling components within root hair cells profoundly impact root hair growth (Cho et al. 2007; Lee and Cho 2008). Moreover, HY1 has been shown to promote lateral root emergence by inducing HY5 and HYH, consequently modulating auxin homeostasis through the regulation of polar auxin transporter localization, including PIN1, PIN2, and AUX1 transporters (Duan et al. 2021).

HY5 promotes lateral root primordia abundance by increasing the local accumulation of auxin at the lateral root primordia (Zhang et al. 2019). Recently, it was shown that HY5 is required for maintaining IAA levels in the roots of Arabidopsis, and loss of function of HY5 results in a decrease of IAA levels in hy5 mutant roots (Burko et al. 2020; Gaillochet et al. 2020). According to Lee et al. (2021), HY5 differently regulates the auxin-responsive genes IAA19, SAUR40, and SAUR77 in shoot and root to promote root thermomorphogenesis. In our study, these findings are supported by ChIP-qPCR, RNA-seq, and RT-qPCR studies. IAA quantification and relative expression analysis of auxin pathway genes in roots of hy5, hy5/HY5OX, WT, cop1-4 suggested that loss of HY5 function results in a reduction of auxin activity (Fig. 4, A and B, Supplementary Fig. S4). PIN2 is involved in the supply of auxin from the root tip to the root hair differentiation zone. So, the loss of function of PIN2 leads to the suppression of root hair growth because it affects the transport of auxin from the root tip to the differentiation zone of root hair (Luschnig et al. 1998; Müller et al. 1998). PIN3 is involved in root hair growth and lateral root development (Lee and Cho 2006; Chen et al. 2015). HY5 alters root auxin accumulation by modulating plasma membrane abundance of PIN2 intracellular auxin transporter (Laxmi et al. 2008).

Our grafting experiments revealed that the expression of auxin reporter at the root tip is almost negligible in roots of hy5 mutant compared to WT, hy5/HY5OX, cop1-4 (Fig. 4, C and D). Auxins have a significant role in root hair formation by initiating root hair developmental genes such as RSL2, RSL4, and EXPA7 (Cho and Cosgrove 2002; Mangano et al. 2017). Accumulation of auxin in the root cap induces root hair formation by activation of ARF19, which leads to the induction of expression of RSL4 and RSL2 (Bhosale et al. 2018). The significant downregulation of root hair developmental genes (RSL2, RSL4, EXPA7) in the hy5 mutant root further confirmed that the aberrant root hair phenotype in the hy5 mutant is caused by the low levels of auxin (Fig. 5, A to C, Supplementary Fig. S4). However, the concept that HY5 positively regulates the expression of root hair developmental genes is supported by the lack of a noticeable change in the expression of these genes in the roots of dark-grown WT and hy5 mutants (Figure 5, A to C). In vitro EMSA and ChIP-qPCR proved the interaction between HY5 protein and promoters of root hair developmental genes (Fig. 5, D to I, Supplementary Fig. S5, A to C). Auxin can induce root hair even in root hairless mutant rhd6 by activating the RSL4 (Yi et al. 2010). However, the aberrant root hair phenotype in the hy5mutant is not restored completely even after the application of an external auxin source (Fig. 6, Supplementary Fig. S6). In general, the external application of auxin increases the expression of RSL2, RSL4, and EXPA7 genes in Arabidopsis. The question arises: why did auxin-treated hy5 mutants not restore the root hair phenotype? The low levels of root hair development genes in the hy5 mutant indicate the importance of HY5 in modulating root hair development genes together with auxin. This implies that HY5 controls root hair development by cooperating with the auxin signaling pathway and root hair development genes. Further evidence that PIN2 and PIN3 are required for root hair development was obtained from the root hair phenotypes of PIN (PINIOID) protein mutants (pin2 and pin3) (Supplementary Fig. S8). After grafting with the scion of the hy5 mutant, root hair regeneration failed in the roots of pin2 and pin3 mutants, demonstrating the importance of HY5 in controlling the components of auxin that regulate root hair development.

In light of these results and analyses, it becomes apparent that the light-responsive HY5 transcription factor is required for the regulation of auxin-mediated root hair growth and development. Our study illuminates the intricate interplay between light, auxin accumulation, and gene pathways associated with miRNA cell walls, crucial for maintaining the appropriate length of root hair.

Materials and methods

Plant materials and growth conditions

WT Arabidopsis (A. thaliana) seeds used in this study were Columbia (Col-0) ecotypes. The mutants cop1-4, hy5 and hy5/HY5OX (hy5/35S:HA-HY5), used were previously described (McNellis et al. 1994; Oyama et al. 1997; Bhatia et al. 2018, 2021). The seeds were surface-sterilized and plated on half-strength Murashige and Skoog (MS) medium (Sigma) supplemented with 1% sucrose. After stratification for 3 days at 4 °C in the dark, the plates were transferred to a growth chamber (Conviron-CG72, Canada) maintained under a long-day photoperiod cycle (16-h light, 8-h dark) and constant temperature (22 °C) for 5 days. As a source of visible light, white cool fluorescent tubes (Philips F54T5/841/HO) with a light intensity of 150 to 180 μmol m⁻²s⁻¹ were used. For the complete darkness treatment, the plates were covered in three layers of aluminum foil and harvested in dim green light.

Plasmid construction and Arabidopsis transformation

The AtmiR397b gene was amplified and cloned into a pBI121 vector using the XbaI and SacI restriction sites to develop a 35S:miR397b construct. Using Agrobacterium-mediated floral dip, the 35S:miR397b construct was transformed into WT Arabidopsis (Col-0). The oligonucleotide primers used to generate overexpression lines are provided in Supplementary Table S1.

CRISPR/Cas 9-mediated genome editing of miR397b

For the editing of miR397b, CRISPR/Cas9 system was used in A. thaliana; gRNAs for miR397b were designed. Both the gRNAs were cloned into the binary vector pHSE401 using the BsaI restriction site (Sharma et al. 2020; Badola et al. 2022). The pHSE401 vectors having gRNAs were transformed into Agrobacterium (strain GV3101) using the freeze–thaw method, and transformation was done by floral dipping methods in Col-0 (Arabidopsis) plants (Clough and Bent 1998). The T0 seeds were collected and screened on half-strength MS plates supplemented with hygromycin (20 mg/L). Positive plants were transferred to soilrite for the maturation. Genomic DNA from the leaves was isolated using the GenElute Plant Genomic DNA Miniprep Kit (Sigma). Regions around the target sites were amplified for the detection of mutations. PCR products were cloned in the cloning vector pTZ57R/T and sequenced using M13F and M13R primers. Mutated plants were grown for further generation to become homozygous and used for study.

Measurement of root growth parameters

To measure the length of root hairs, root hairs located 5 mm from the root tip of 4- to 7-d-old grown WT, miR397bOX homozygous lines, hy5, hy5/HY5OX, and cop1-4 seedlings were photographed under a Leica microscope (LAS version 4.12.0, Leica Microsystems). The digital images were used directly for root hair length measurement using ImageJ software https://imagej.nih.gov/ij/. We repeated all the experiments with at least three replicates of 8 to 15 seedlings.

RLM-RACE analysis

To map the cleavage sites of the candidate targets of miR397b in planta, modified 5′ RLM-RACE was performed using a First Choice RLM-RACE Kit (Invitrogen). Total RNA was extracted from the 5-d-old seedlings of the miR397bOX line and ligated directly to an RNA oligonucleotide adapter without Calf Intestine Alkaline Phosphatase (CIP) and Tobacco Acid Pyrophosphatase (TAP) treatment. cDNA synthesis was performed using oligo(dT) primers using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Applied Biosystems). RACE and nested-RACE PCR were subsequently carried out with adaptor-specific forward primers and gene-specific reverse primers. The PCR products were cloned into the pTZ57R/T subcloning vector (Fermentas) and sequenced to locate the miRNA cleavage sites.

RNA isolation and expression analysis

Total RNA was isolated using a Spectrum Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer's instructions. For cDNA synthesis, total RNA was treated with Thermo Scientific DNase (Ambion), and 1 µg was reverse-transcribed using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Applied Biosystems) according to the manufacturer's instructions. The cDNA was diluted 10 times with nuclease-free water, and 2 µL of cDNA was used as a template for quantitative PCR, which was performed using Fast SYBR Green Mix (Applied Biosystems) in a Fast 7500 Thermal Cycler instrument (Applied Biosystems). Expression was normalized using the tubulin housekeeping gene and analyzed through the comparative ΔΔ^CT method (Livak and Schmittgen 2001). For quantification of mature miRNA, TaqMan microRNA assay was performed. cDNA was synthesized using microRNA-specific primers, and TaqMan Reverse Transcription Synthesis Kit (Thermo Scientific) followed by qPCR was performed using TaqMan probes and TaqMan Universal Master Mix with no UNG (Thermo Scientific). The oligonucleotide primers used to study the expression of different genes were designed using the Primer Express 3.0.1 tool (Applied Biosystems). Information is provided in Supplementary Table S1.

Arabidopsis grafting experiments

For homo- or hetero-grafting between DR5Pro/WT, hy5, hy5/HY5OX, and cop1-4, hypocotyl grafting was performed by using seedlings grown for 4 days on media. Cotyledons were excised from the seedlings without disturbing the shoot apex, and then, approximately 180° cuts were made using a sterilized scalpel, between scion and stock. Desired scion and stock were placed on each other with different combinations and allowed to grow for 12 days in a growth chamber before GUS histochemical assay was performed (Sharma et al. 2022; Vanderstraeten et al. 2022). For homo- or hetero-grafting between pin2, pin3/WT, hy5, and hy5/HY5OX, cop 1-4 followed the same method. After 12 days, phenotypic analysis of root hairs was analyzed under a phase-contrast microscope.

GUS expression analysis

GUS staining was performed using a previously described method (Jefferson 1989). Briefly, grafted seedlings were immersed in a solution containing 100 mm sodium phosphate buffer (pH 7.2), 10 mm EDTA, 0.1% (v/v) Triton X-100, 2 mm potassium ferricyanide, 2 mm potassium ferrocyanide, and 1 mg mL⁻¹ 5-bromo-4-chloro-3-indolyl-β-D-glucuronide at 37 °C for 12 h. Chlorophyll was removed by incubation and multiple washes using 70% (v/v) ethanol. The seedlings were placed on a slide for observations under a Leica microscope (LAS version 4.12.0, Leica Microsystems) for GUS staining.

LC–MS/MS analysis

Auxin analysis was performed on three biological replicates of 7-d-old seedlings of WT, hy5, hy5/HY5OX, and cop1-4 using standard method (Pan et al. 2010). Briefly, by using a mortar and pestle, seedlings were ground into fine powder and then extraction solution (80% [v/v] methanol) was added to the tube followed by keeping tubes in a shaker at a speed of 100 rpm for 30 min at a temperature of 4 °C. The samples were transferred into a microcentrifuge set at 4 °C, 12,000rpm for 15 min. The collected aqueous phase was evaporated by using a nitrogen evaporator. The concentrated aqueous phase was dissolved in 80% (v/v) methanol. The Ultra Performance Liquid Chromatography (UPLC) analysis was performed on an instrument of the Agilent 1290 series (Agilent Technologies, Santa Carla, CA, USA), composed of a binary pump (G7120A), an autosampler (G7129A), and a column oven (G7130A). The UPLC separation was accomplished on a Zorbax Eclipse Plus C18 Rapid Resolution HD 1.8 µm 2.1 × 150 mm (Agilent Technologies, Santa Carla, CA, USA) operated at 40 °C. Gradient elution was achieved using two solvents: 0.1% (v/v) formic acid aqueous solution (A) and 0.1% (v/v) formic acid in acetonitrile (B) at a flow rate of 0.2 mL/min. The 15-min UPLC gradient elution program was as follows: (i) 20%, from 0 to 3 min (B); (ii) 30%, from 3 to 4.5 min (B); (iii) 50%, from 4.5 to 6 min (B), 70%, from 6 to 8.5 min (B), 90%, from 8.5 to 10 min (B), 50%, from 10 to 12 min (B), and 10%, from 12 to 15 min (B) of total run time; the injection volume was 2 µL. The MS analyses were performed on a QTOF-MS instrument of the Agilent 6545 series (G6545A), connected with an Agilent 1290 UPLC (Agilent Technologies, Santa Clara, CA, USA) through a dual AJS ESI interface. Nitrogen was used as the drying and collision gas in the ESI source. The ion source parameters were as follows: a drying gas flow rate, 10 mL/min; a heated capillary temperature, 330 °C; a nebulizer pressure, 35 psi; and VCap, fragmentor, skimmer, and octopole RF peak voltages set at 4000, 180, 45, and 750 V, respectively. The detection was carried out in positive electrospray ionization mode, and the spectra were recorded by MS scanning in the m/z 100 to 1700. The MS/MS analyses were carried out with a collision energy of 30 eV. MassHunter software version B.07.00 (Agilent Technology) was used to control the LC–MS/MS system, data acquisition, and processing. MS/MS production of quantitative LC–MS/MS positive transition considered m/z = 130.0655.

Electrophoretic mobility shift assay

The second-generation DIG Gel Shift EMSA kit (Roche, USA) was used to label the probes with digoxigenin as per the manufacturer's instructions. Labeled probes were incubated at 21 °C for 30 min in binding buffer (100 mm HEPES [pH 7.6], 5 mm EDTA, 50 mm (NH₄)₂SO₄, 5 mm DTT, Tween 20, 1% [w/v], 150 mm KCl) with or without recombinant protein. Unlabeled probes were added to the reaction solution in increasing concentrations to test specific binding. The binding reaction was resolved on a 6% polyacrylamide gel in 0.5X TBE (pH 8.0) buffer and semi-dry blotted (Transblot, BIO-RAD, USA) onto a positively charged nylon membrane (BrightStar, Invitrogen, USA) followed by UV cross-linking. The membrane was finally incubated with Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) chemiluminescent solution and exposed to X-ray blue film (Retina, India).

Chromatin immunoprecipitation

ChIP was performed on 10-d-old seedlings. Seedling tissues (2 g) were cross-linked with 50 mL of 1% formaldehyde in a vacuum for 20 min. A total of 2.5 mL of 2 m glycine was added to stop the cross-linking. After rinsing seedlings with water, tissues were ground in liquid N₂, resuspended in 20 mL of extraction buffer I (EB I) (0.4 m sucrose, 10 mm Tris–HCl, pH 8, 10 mm MgCl₂, 5 mm beta-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride [PMSF], and 13 protease inhibitor; Sigma), and then filtered through a cell strainer (Corning). The filtrate was centrifuged at 4,000 rpm at 4 °C for 30 min. The pellet was resuspended in 1 mL of extraction buffer II (EB II) (0.25 m sucrose, 10 mm Tris–HCl, pH 8, 10 mm MgCl₂, 1% Triton X-100, 5 mm beta-mercaptoethanol, 0.1 mm PMSF, and protease inhibitor) and centrifuged at 14,000 rpm and 4 °C for 10 min. The pellet was resuspended in 300 mL of extraction buffer III (EB III) (1.7 m sucrose, 10 mm Tris–HCl, pH 8, 0.15% Triton X-100, 2 mm MgCl₂, 5 mm beta-mercaptoethanol, 0.1 mm PMSF, and 13 protease inhibitor), loaded on top of an equal amount of clean EB III, and then centrifuged at 14,000 rpm for 1 h. The crude nuclear pellet was resuspended in nuclear lysis buffer (50 mm Tris–HCl, pH 8.0, 10 mm EDTA, 1% SDS, and complete protease inhibitor; Roche) and sonicated with a Branson sonifier (VWR) to achieve an average fragment size of 0.3 to 0.8 kb. The sonicated chromatin was centrifuged, and the insoluble pellet was discarded. The soluble chromatin solution was diluted 10-fold with ChIP dilution buffer (1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris–HCl, pH 8.0, and 167 mm NaCl), and then, after preclearing with protein-A Sepharose beads (Sigma-Aldrich), 40 µL of HA tag-specific monoclonal antibody (Roche) was added to 1 mL of chromatin solution and incubated overnight at 4 °C. The immunocomplexes were extracted by incubating with 100 mL of 50% protein-A Sepharose beads for 1 h at 48 °C. After several washes, immunocomplex was eluted twice from the beads with 250 mL of elution buffer (1% SDS and 0.1 m NaHCO₃) and then reverse cross-linked with a final concentration of 200 mm NaCl at 65 °C for 12 h (overnight). After removing all proteins by treating with proteinase K, DNA was purified by phenol–chloroform extraction, followed by ethanol precipitation. The pellet was resuspended in 50 mL of 0.13 TE (10 mm Tris–EDTA, pH 7.5) with RNase A (0.1 mg/mL) and used for probe synthesis or PCR analysis.

Statistical analysis

Data are plotted as means ± SD with error bars as SD. The statistical tests and n numbers, including sample sizes or biological replications, are described in the figure legends. All the statistical analyses were performed using two-tailed Student's t-tests using GraphPad Prism version 8.4.3 software (ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001). All the experiments were repeated at least three times independently, with similar results.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: AtHY5 (AT5G11260), AtCOP9 (AT4G14110), AtmiR397b (AT4G13555), AtPIN2 (AT5G57090), AtPIN3 (AT1G70940), AtRRA1 (AT1G75120), AtRRA2 (AT1G75110), AtRSL2 (AT4G33880), AtRSL4 (AT1G27740), and AtEXPA7 (AT1G12560).

Acknowledgments

The authors thank Prof. Ute Hoecker for providing hy5-215, cop1-4 mutant seeds, Dr. A. P. Sane for providing pin2 and pin3 mutant seeds, Q.-J. Chen at China Agriculture University for the pHSE401 vector, and N.M. Sabiq (ICAR-Central Marine Fisheries Research Institute [CMFRI]) for help in compiling the microscopic figures. CIMAP publication number is CIMAP/PUB/2024/61.

Author contributions

S.R.G., C.B., and P.K.T. designed the study. S.R.G. and A.S. carried out the experiments. S.R.G., C.B., A.S., and P.K.T. analyzed and wrote the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Generation of miR397b^CR edited lines by using CRSIPR/Cas9 method.

Supplementary Figure S2. IAA standard curve for LC–MS/MS analysis.

Supplementary Figure S3. Relative expression of auxin pathway genes.

Supplementary Figure S4. Relative expression of root hair developmental genes.

Supplementary Figure S5. EMSA interaction showing HY5 binding with root hair developmental genes.

Supplementary Figure S6. Graphical representation of root hair number in auxin-treated seedlings.

Supplementary Figure S7. Measurement of lateral root number in auxin-treated seedlings.

Supplementary Figure S8. Representation of root hair phenotype of pin2 and pin3 mutants.

Supplementary Table S1. Oligonucleotides used for the development of constructs and expression analysis.

Funding

This research was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, in the form of NCP project no. MLP006. P.K.T. also acknowledges the Science and Engineering Research Board (SERB), New Delhi, for JC Bose National Fellowship (JCB/2021/000036). S.R.G. acknowledges the University Grant Commission, New Delhi, for Senior Research Fellowship, A.S. acknowledges the DST/INSPIRE faculty fellowship, and C.B. acknowledges the M.K. Bhan Fellowship, Department of Biotechnology, Delhi, India, for fellowship.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• LAS Gramene: AT1G55580

• LAS Araport: AT1G55580

• PIN3 Gramene: AT1G70940

• PIN3 Araport: AT1G70940

• SDS Gramene: AT1G14750

• SDS Araport: AT1G14750

• ARF19 Gramene: AT1G19220

• ARF19 Araport: AT1G19220

• AUX1 Gramene: AT2G38120

• AUX1 Araport: AT2G38120

• HYH Gramene: AT3G17609

• HYH Araport: AT3G17609

• PIN1 Gramene: AT1G73590

• PIN1 Araport: AT1G73590

• RRA1 Gramene: AT1G75120

• RRA1 Araport: AT1G75120

• RRA2 Gramene: AT1G75110

• RRA2 Araport: AT1G75110

• AtEXPA7 Gramene: AT1G12560

• AtEXPA7 Araport: AT1G12560

• HY5 Gramene: AT5G11260

• HY5 Araport: AT5G11260

• IAA19 Gramene: AT3G15540

• IAA19 Araport: AT3G15540

• EDTA Gramene: Ethylenediamine tetraacetic acid

• EDTA Araport: Ethylenediamine tetraacetic acid

• HEPES CHEBI: CHEBI:46756

• RHD6 Gramene: ROOT HAIR DEFECTIVE SIX

• RHD6 Araport: ROOT HAIR DEFECTIVE SIX

• AtHY5 Gramene: At5g11260

• AtHY5 Araport: At5g11260

References

Author notes