PGA37 Overexpression Promotes Chloroplast Development in Arabidopsis Roots Through Direct Transcriptional Activation of GLK2, ARR13, and ARR21

TL;DR

This study shows how a specific plant protein, PGA37, helps develop chloroplasts in roots by activating other key genes.

Contribution

The study identifies PGA37 as a novel regulator of root chloroplast development through direct activation of GLK2, ARR13, and ARR21.

Findings

PGA37 overexpression causes root-greening by promoting chloroplast development.

PGA37 directly activates GLK2, ARR13, and ARR21, which are involved in chloroplast biogenesis and cytokinin signaling.

GLK2 overexpression replicates the root-greening effect, while its mutation reduces chlorophyll content.

Abstract

Chloroplast biogenesis and development are essential processes in plants, profoundly influencing their growth, survival, and productivity. However, the transcription factors controlling chloroplast development, especially in roots, are poorly characterized. Here, we demonstrate that the ectopic expression of the seed-specific transcription factor Plant Growth Regulator 37 (PGA37) promotes chloroplast development in roots, causing root-greening. Using a steroid-inducible gene expression system and RNA-Seq, we identified 97 potential PGA37 target genes. Notably, PGA37 directly activates the transcription factor GOLDEN2-LIKE (GLK2), which governs chloroplast biogenesis. An overexpression of GLK2 replicated the root-greening phenotype observed in PGA37-overexpressing plants, while GLK2 mutation significantly reduced chlorophyll content and suppressed root-greening in PGA37-overexpressing seedlings. Furthermore, PGA37 directly binds to the promoters of type-B response regulators ARR13 and ARR21, thereby activating the cytokinin signaling pathway. Mutations in these regulators partially diminished chlorophyll accumulation in PGA37-overexpressing seedlings, suggesting that PGA37-regulated chloroplast development is partially mediated by the cytokinin signaling through ARR13 and ARR21. Taken together, we propose that PGA37 orchestrates chloroplast development by coordinately regulating transcription factors from various families, including GLK2, ARR13, and ARR21, positioning it as a key regulator of chloroplast development.

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Full text

1. Introduction

Chloroplasts can develop directly from the undeveloped proplastids or from etioplasts; their intermediate structures form in the absence of light [1]. During this process, thylakoids are formed and stacked into defined grana. Thylakoids are crucial structures in chloroplasts, which serve as the primary sites for the light-dependent reactions of photosynthesis. Another hallmark of chloroplast generation is chlorophyll biosynthesis. Chlorophyll is synthesized from glutamate, which is converted to 5-aminolevulinic acid (ALA) by the sequential reactions of Glu-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate 1-semialdehyde aminotransferase (GSAT) [2,3]. Among these enzymes, GluTR represents the first rate-limiting step in ALA synthesis. In Arabidopsis, GluTR enzymes are encoded by three nuclear genes, HEMA1, HEMA2, and HEMA3. Notably, HEMA1 is the predominant gene, and its downregulation leads to chlorophyll deficiency, which negatively affects plant growth and development [4].

Light is a crucial environmental factor that regulates chloroplast development and chlorophyll biosynthesis in plants [5]. Light-dependent pathways involve photoreceptors, such as phytochromes and cryptochromes, which perceive different wavelengths of light and transmit signals that modulate gene expression [6]. Among the downstream regulators, the bZIP transcription factor ELONGATED HYPOCOTYL 5 (HY5) acts as a central regulator of chloroplast development by integrating signals from these photoreceptors [7]. This integration is vital for the coordinated expression of genes essential for chloroplast biogenesis and function. A recent study demonstrated that HY5 directly activates the expression of two GOLDEN2-LIKE (GLK) family transcription factors, GLK1 and GLK2, which are key regulators of chloroplast biogenesis [8,9]. These GLKs function synergistically with MYB-related transcription factors to enhance the expression of several nucleus-encoded genes critical for photosynthesis, particularly those involved in chlorophyll biosynthesis and light-harvesting capabilities [10,11]. The double mutant glk1 glk2 exhibits a distinct pale-green phenotype and a significant reduction in thylakoid formation, underscoring the critical roles of GLK1 and GLK2 in chlorophyll biosynthesis and chloroplast development [12]. Conversely, an overexpression of either GLK1 or GLK2 induces chloroplast development in non-green tissues, suggesting their potential use in biotechnological applications aimed at enhancing photosynthetic efficiency in crop plants [13,14].

Phytohormones, particularly cytokinins, play an essential role in chloroplast biogenesis and function [15]. The involvement of cytokinins in delaying chlorophyll degradation was recognized soon after their identification as plant growth regulators [16]. Over the past two decades, significant insights into the molecular pathways governing cytokinin-mediated chloroplast development have emerged. In Arabidopsis, cytokinin is perceived by three histidine kinases: ARABIDOPSIS HIS KINASE2 (AHK2), AHK3, and CRE1/AHK4, which function as cytokinin receptors. Upon cytokinin binding, these receptors transduce signals to type-B Arabidopsis response regulators (type-B ARRs) through histidine phosphotransfer proteins (AHPs) [17]. Type-B ARRs subsequently activate the transcription of downstream target genes that regulate chloroplast development [18]. Among these receptors, AHK3 plays a pivotal role in mediating the effects of cytokinins on chloroplast development and chlorophyll accumulation. Mutations in AHK3 result in an approximately 25% reduction in chlorophyll content compared to wild-type plants, while mutations in AHK2 have a negligible impact and AHK4 mutations exhibit no effect on chlorophyll levels [19]. Furthermore, a significant reduction in chlorophyll content has been observed in the triple type-B ARR mutant arr1 arr10 arr12 [18], indicating that the target genes of these type-B ARRs are essential for chloroplast development. Notably, ARR10 and ARR12 specifically bind to the promoters of HEMA1 and LIGHT HARVESTING COMPLEX PHOTOSYSTEM II SUBUNIT6 (LHCB6) [2]. HEMA1 and LHCB6 encode glutamyl-tRNA reductase and Glu-1-semialdehyde aminotransferase, respectively, which catalyze the initial rate-limiting steps of chlorophyll synthesis.

Chloroplast differentiation in roots is mechanistically different from that in aerial tissues; however, the regulatory network governing root chloroplast development can be equally complex, comprising multiple transcription factors. Kobayashi et al. [20] have reported that the B-GATA transcription factor GNC-LIKE/CYTOKININ-RESPONSIVE GATA1 (GNL) is significantly upregulated in detached roots mediated by ARR1 and ARR12. Although GNL does not directly participate in chlorophyll synthesis, its overexpression promotes ectopic chloroplast development, while a loss of GNL function results in reduced chlorophyll accumulation. Collectively, these findings suggest that ARR1, ARR10, and ARR12 functionally overlap and act as key regulators of cytokinin signaling during chloroplast development and chlorophyll accumulation. In Arabidopsis, there are 11 type-B ARRs that can be categorized into three subfamilies based on phylogenetic analysis. However, other than ARR1, ARR10, and ARR12, the roles of the remaining type-B ARRs in chloroplast development remain largely unexplored.

During embryogenesis, the formation of chloroplasts from proplastids is as critical as their development in vegetative organs. However, the regulatory mechanisms of chloroplast development and chlorophyll biosynthesis in reproductive organs remain poorly understood. In a previous study, we reported that overexpression of the seed-specific R2R3-MYB transcription factor PGA37 induces the formation of somatic embryos in Arabidopsis [21]. In this study, we further investigated the role of PGA37 in chloroplast development, emphasizing its essential function in regulating chlorophyll accumulation in seeds and chloroplast biogenesis in roots through the direct regulation of GLK2, ARR13, and ARR21.

2. Results

2.1. Ectopic Expression of PGA37 Induces Chloroplast Development in Roots

In higher plant roots, chloroplast development is typically suppressed even under light conditions, resulting in roots that are generally white or yellowish (Figure 1A). However, the roots of the plant growth activator 37 (pga37) mutant exhibit a distinct light-green phenotype, indicating the formation of chloroplasts in pga37 roots (Figure 1A). The pga37 is a gain-of-function mutant in which the ectopic expression of the seed-specific PGA37 gene can be induced by estradiol [21]. Quantitative analysis revealed that the total chlorophyll content in roots of pga37 seedlings grown on an inductive medium with estradiol is approximately 3.9 times higher than that of roots on non-inductive medium (Figure 1B). Further chlorophyll autofluorescence analysis revealed that chlorophyll was predominantly accumulated in the thickened stele of the pga37 primary root (Figure 1C,D). Additionally, chlorophyll accumulation was also observed in the outer cell layers, endodermis, and cortex (Figure 1C,D). A similar distribution pattern of chlorophyll-containing cells was noted in the roots of GLK1 or GLK2 overexpression lines [13]. To ascertain whether chloroplasts developed in the green root of the pga37 or not, an ultrastructural analysis of pga37 roots was conducted using transmission electron microscopy. In the absence of estradiol, pga37 seedlings exhibited poorly developed thylakoid membrane networks in root plastids (Figure 1E). Conversely, root plastids exposed to 10 μM estradiol displayed well-formed thylakoid membranes and grana structures (Figure 1F).

To further confirm that the green root phenotype was attributable to the gain-of-function mutation in PGA37, we examined the phenotype of pER10-PGA37 transgenic seedlings. Given that the growth and development of pER10-PGA37 transgenic plants were completely inhibited at higher concentrations of the inducer, we opted to germinate the seeds on MS medium supplemented with 0.1 μM 17-β-estradiol. The green root phenotype observed in pga37 gain-of-function mutant was successfully replicated in the pER10-PGA37 transgenic plants (Figure 1G). Furthermore, the chlorophyll distribution pattern in the roots of pER10-PGA37 transgenic seedlings closely mirrored that observed in the roots of pga37 mutants (Figure 1H,I), reinforcing the causal link between PGA37 overexpression and the observed chlorophyll accumulation. (Figure 1H,I).

2.2. Light Enhances the Regulation of Chloroplast Development by PGA37

Previously, Kobayashi et al. [22] reported that light signaling was required for the greening of detached roots. To investigate whether chloroplast development in the pga37 mutant depends on light, detached pga37 roots were cultured on MS medium containing 10 μM 17-β-estradiol under both light and dark conditions. The results demonstrated that the application of estradiol induced a greening phenotype in the pga37 mutant roots under both conditions (Figure 2A). However, it was observed that the roots accumulated significantly more chlorophyll when estradiol was applied under light conditions compared to dark conditions (Figure 2A,B). To further investigate the influence of light on PGA37, we examined the effect of PGA37 overexpression on the expression of HY5, a key regulatory gene in the light signaling pathway. Under dark conditions, PGA37 overexpression had no significant impact on HY5 expression (Figure 2C). However, under light conditions, HY5 expression was notably inhibited by PGA37 (Figure 2C). These findings suggest that PGA37 requires light to maximize its activities in root greening and that it also influences the expression of HY5.

2.3. Genome-Wide Identification of the Potential Targets of PGA37 Using a Glucocorticoid-Inducible System

To further elucidate the role of PGA37 in chloroplast development, we conducted RNA-Seq analysis to identify the direct target genes of PGA37, utilizing a glucocorticoid receptor (GR)-based inducible system 35S::PGA37-GR (Figure 3A). In this system, the activation of PGA37 target genes was independent of protein synthesis when the 35S::PGA37-GR transgenic seedlings were treated simultaneously with dexamethasone (DEX) and cycloheximide (CHX) [23]. Therefore, this suggests a direct relationship between the treatment and the activation of PGA37 target genes. In the absence of DEX induction, the 35S::PGA37-GR transgenic seedlings displayed phenotypes that were indistinguishable from the wild-type. However, upon DEX induction, the phenotypes of pga37 mutant and pER10-PGA37 transgenic seedlings were recapitulated in 35S::PGA37-GR transgenic plants, including root-greening, the distribution pattern of chlorophyll-containing cells, the retarded growth and development of the transgenic seedlings, and the somatic embryo formation of the root explants (Figure 3B–E and Figure S1). Notably, our previous research suggested that PGA37 acts upstream from LEC1 to regulate the vegetative-to-embryonic transition [21]. Supporting this, we observed that LEC1 expression was rapidly induced by DEX in the 35S::PGA37-GR transgenic seedlings (Figure 3F). Collectively, these results demonstrate that the PGA37-GR fusion protein retains its biological activity and effectively modulates the expression of its target genes upon DEX induction, thereby confirming its role in promoting chloroplast development.

The 35S::PGA37-GR transgenic seedlings were grown under normal conditions for 2 weeks and then treated with DEX and CHX. Subsequently, RNA-Seq was employed to analyze gene expression patterns. The RNA-Seq data revealed 97 differentially expressed genes, with 90 upregulated and 7 downregulated (Table S1). Since the secondary transcriptional regulation downstream of PGA37 was prevented by CHX, these genes are potential direct targets of PGA37. Most of the differentially expressed genes are involved in several biological processes, including porphyrin metabolism, fatty acid biosynthesis, fatty acid metabolism, and cytokinin signal transduction (Figures S2 and S3). Consistent with our previous findings that an overexpression of PGA37 leads to lipid accumulation [21], we observed a significant upregulation of AtDES3, a key gene involved in oleic acid synthesis, in the 35S::PGA37-GR transgenic seedlings following DEX treatment (Table S1). Further analysis revealed that 88 of the 90 upregulated genes and all downregulated genes contained at least one MYB binding sequence in their 1.5 kb upstream regions (Figure S4). Among the two exceptions, AT5G57480 encodes a P-loop containing nucleoside triphosphate hydrolases superfamily protein, while AT2G13910 is a pseudogene and its 1.5 kb upstream region could not be obtained.

2.4. PGA37 Induces Chloroplast Development by Directly Regulating the Expression of GLK2 but Not GLK1

Among the 97 potential PGA37 target genes, we found that the GLK2 gene showed a significant upregulation in seedlings of 35S::PGA37-GR upon DEX induction (Figure 4A and Table S1). Therefore, the rapid activation of GLK2 by PGA37 suggests a direct regulatory mechanism, likely involving the interaction of PGA37 with the GLK2 promoter. To further substantiate the direct interaction between PGA37 and the GLK2 promoter, we conducted biolayer interferometry (BLI) assays. As depicted in Figure 4B, PGA37 exhibited a strong affinity for the GLK2 promoter region, with a dissociation constant (Kd) of 2.32 × 10 ^−7^ M, thereby confirming the direct binding of PGA37 to the GLK2 promoter.

To further investigate the role of PAG37 in chloroplast development, we characterized the phenotype of GLK2 overexpression lines (GLK2ox) and examined the effect of its mutation on chlorophyll accumulation in the roots of 35S::PGA37-GR seedlings. Under normal light conditions, the aerial parts of GLK2ox showed no significant differences compared to the wild type (Figure 4C). However, under high light conditions, GLK2ox accumulates anthocyanins in the aerial parts and exhibited stunted growth and development (Figure 4D). Notably, the roots of GLK2 overexpression seedlings showed a significant increase in chlorophyll accumulation, resulting in a root-greening phenotype similar to that observed in pga37 mutants and pER10-PGA37 transgenic seedlings (Figure 4E,F). Conversely, the root-greening phenotype induced by PGA37 overexpression was suppressed by the mutation of the GLK2 gene (Figure 4G,H). These findings clearly demonstrate that PGA37 induces chloroplast development by promoting the expression of GLK2.

In Arabidopsis, two GLK genes, GLK1 and GLK2, function redundantly to regulate chloroplast development [12,24]. Additionally, an overexpression of GLK1 has been reported to induce a green root phenotype similar to that observed with GLK2 overexpression [13]. Given this, it is possible that GLK1 might also play a role in PGA37-mediated chloroplast development. However, our analysis revealed that GLK1 expression was not induced in PGA37 overexpression seedlings, suggesting that GLK1 is dispensable for the root-greening phenotype observed in 35S::PGA37-GR transgenic lines (Table S1 and Figure S5a). Moreover, BLI assay indicates that PGA37 protein could not bind to the GLK1 promoter (Figure S5b). These findings indicate a specific regulatory role for GLK2 in the PGA37-mediated pathway, underscoring the distinct contribution of GLK2 to chloroplast development under the influence of PGA37.

2.5. PGA37 Promotes Root-Greening Partially Through Cytokinin Signaling Activated by ARR13 and ARR21

We observed a significantly lower chlorophyll content in the roots of GLK2 overexpression (GLK2ox) plants compared to pga37 mutants (Figure 1B and Figure 4F). Moreover, our previous finding indicated that PGA37 can promote the vegetative-to-embryonic transition independently of cytokinin [21]. These findings led us to hypothesize that the root-greening phenotype in pga37 mutants might be at least partially mediated by cytokinin signaling activated by PGA37. Supporting this hypothesis, we found that two type-B ARR genes, ARR13 and ARR21, were significantly induced by DEX in 35S::PGA37-GR transgenic lines (Table S1, Figure 5A,B). Further BLI assays confirm the direct interaction between PGA37 transcription factor and the fragments containing the upstream regulatory sequences near the translational start sites of both ARR13 and ARR21 (Figure 5C,D).

To elucidate the dependency of the PGA37-mediated root-greening phenotype on ARR13 and/or ARR21, we generated 35S::PGA37-GR arr13, 35S::PGA37-GR arr21, and 35S::PGA37-GR arr13 arr21 lines by crossing the arr13 arr21 double mutant with 35S::PGA37-GR transgenic line. Surprisingly, a single mutation in either ARR13 or ARR21 partially suppressed the growth-retardant phenotype caused by PGA37 overexpression (Figure 6A). In contrast, a double mutation in both ARR13 and ARR21 nearly completely suppressed the growth-retardant phenotype. This suggests that the growth-retardant phenotype observed in PGA37 overexpression lines is due to the ectopic expression of ARR13 and ARR21. Interestingly, despite the fact that the single mutation of ARR13 or ARR21 did not affect root chloroplast development in the PGA37 overexpression lines, the chlorophyll content in the roots of 35S:PGA37-GR arr13 arr21 triple mutants was significantly decreased (Figure 6B,C). To gain deeper insight into the role of ARR21 in plant growth and chlorophyll biosynthesis, we analyzed the phenotype of pER8-ARR21 estrogen-inducible overexpression lines. As anticipated, inducible overexpression of ARR21 led to a marked growth and developmental retardation, closely resembling the phenotype observed with cytokinin application (Figure 6D). Under dark conditions, there was no significant difference in chlorophyll content between the roots of pER8-ARR21 transgenic lines treated with 10 μM estradiol and those left untreated (Figure 6E,F). However, under light conditions, the chlorophyll content was significantly higher in estradiol-treated roots compared to untreated roots. Taken together, our results demonstrate that PGA37 activates cytokinin signaling by directly regulating the expression of ARR13 and ARR21, ultimately contributing to the occurrence of ectopic chloroplast development in Arabidopsis.

3. Discussion

Chloroplasts are essential for plant growth and development, as they enable photosynthesis and significantly impact energy production and biomass accumulation, which ultimately influence crop yield and productivity. Therefore, the targeted engineering of this process could significantly contribute to crop improvement and help to sustainably meet global food and bioenergy demands [25]. In this study, we elucidated that an overexpression of PGA37, a seed-specific gene, leads to the ectopic induction of chloroplasts in roots by integrating cross-family transcription factors, including those from families of MYB (PGA37/MYB118), GLK (GLK2), bZIP (HY5), and type-B ARR (ARR13 and ARR21) (Figure 7). Since chloroplasts are typically absent in roots, our findings offer potential for enhancing photosynthesis in conditions where roots are exposed to light, such as in hydroponic or soilless cultivation systems. Furthermore, the homologs of PGA37 may play a more crucial role in epiphytic plants, such as many species within the Orchidaceae family, whose root systems are capable of photosynthesis [26]. This approach could lead to increased yield and productivity under these specific agricultural practices.

In higher plants, the development of chloroplasts in roots is generally suppressed, even when exposed to light [13], making the light-independent chloroplast biogenesis in pga37 roots particularly remarkable. Previous studies, such as those by Kobayashi et al. [22], have shown that light signaling is crucial for chloroplast development in detached roots. However, our results clearly show that PGA37 could induce chloroplast formation in pga37 roots under both light and dark conditions, with a significant increase in chlorophyll content under light conditions (Figure 2A,B). This observation suggests a synergistic interaction between PGA37 and light that enhances chloroplast development, although light is not strictly necessary for the process. The elevated accumulation of chlorophyll observed in the roots of PGA37-overexpressing plants under dark conditions may be due to the activation of GLK2. However, other pathways activated by PGA37, such as the cytokinin signaling pathway, could also contribute to chloroplast induction under dark conditions. Interestingly, we found that the expression of HY5 was significantly repressed by PGA37 under light conditions (Figure 2C). It has been reported that cytokinin can increase HY5 protein accumulation by reducing its CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1)-dependent degradation [27]. We hypothesize that the activation of the cytokinin signaling pathway by ARR13 and ARR21 might lead to an increase in the HY5 protein levels, resulting in a negative feedback regulation of HY5 gene expression. However, we cannot rule out the possibility that PGA37 may interact with HY5, a light-induced protein, which could also, through negative feedback, regulate the expression of HY5. In contrast to *PGA37′*s role in promoting chloroplast development in roots, we observed that the 35S::PGA37GR and pER10-PGA37 transgenic seedlings displayed yellowish leaves when cultivated on a medium with a relative high concentration of inducer (Figure 3B, see also reference [21] (Wang et al., 2009). We hypothesize that this reduction in leaf chlorophyll is related to the expression levels of PGA37. At lower expression levels, PGA37 promotes chloroplast development. However, at excessively high expression levels, it may activate the synthesis of other products through its target genes, which, at elevated levels, could become toxic and result in chlorophyll degradation in leaves. Furthermore, the basal levels of these potentially toxic products are inherently lower in roots compared to leaves, causing a reduced-chlorophyll phenotype to appear in leaves first, where these products are naturally more abundant. In addition, the excessive production of certain intermediates or chlorophyll itself may trigger the feedback inhibition of the chlorophyll biosynthetic pathway, leading to the pale-yellow phenotype. Further experimental evidence will be required to validate this hypothesis.

The genome-wide identification of potential PGA37 target genes using a glucocorticoid-inducible system has provided valuable insights into the downstream regulatory network controlled by PGA37. RNA-Seq analysis identified 97 differentially expressed genes, with most being upregulated, indicating that PGA37 predominantly acts as a transcriptional activator (Table S1). Among these differentially expressed genes, GLK2 was identified as a direct target, showing rapid upregulation upon the induction of PGA37 expression (Figure 4). The role of GLK2 in chloroplast development is further supported by the phenotypic analysis of GLK2 overexpression lines, which exhibited a root-greening phenotype similar to that of pga37 mutants (Figure 4E,F). The suppression of this phenotype by GLK2 mutation in PGA37 overexpression lines underscores *GLK2′*s important role in this pathway (Figure 4G,H). The lack of GLK1 induction and the inability of PGA37 binding to the GLK1 promoter highlight the specific and non-redundant function of GLK2 in PGA37-mediated chloroplast development, suggesting that GLK1 is dispensable in this context (Figure S5). Moreover, GLK1 is scarcely expressed in Arabidopsis roots, while GLK2, but not GLK1, was upregulated in roots treated with cytokinin [22]. These findings consistently support the crucial role of GLK2 in chloroplast development mediated by PGA37. In contrast, recent findings by Frangedakis et al. [11] revealed that two RR-MYBs, AtMYBS1 and AtMYBS2, can bind to the promoter of GLK1 but not GLK2. This suggests a distinct regulatory mechanism involving RR-MYBs and GLK1 that may complement or diverge from the pathways mediated by PGA37 and GLK2. These insights broaden our understanding of the complex regulatory networks governing chloroplast development.

In Arabidopsis, there are 11 type-B ARRs categorized into three distinct subfamilies [28]. Previous research by Cortleven et al. [2] has identified three type-B ARRs from subfamily I—ARR1, ARR10, and ARR12—as critical players in chloroplast development. Notably, ARR1 and ARR12 have been shown to be essential for chloroplast development specifically in detached roots [20]. In our study, we discovered that ARR13 and ARR21, both from subfamily II, are direct targets of PGA37 (Figure 5). Furthermore, the significant reduction in chlorophyll content in the roots of 35S::PGA37-GR arr13 arr21 triple mutants provides compelling evidence that cytokinin signaling, mediated by ARR13 and ARR21, plays a role in the ectopic development of chloroplasts in roots (Figure 6). This finding underscores the complex regulatory mechanisms orchestrated by cytokinin signaling in chloroplast development.

It is noteworthy that ARR13 and ARR21, like PGA37, are primarily expressed in reproductive organs [21,29,30,31]. Further investigation is needed to determine whether ARR13 and ARR21 contribute to chloroplast development in detached roots independently of PGA37. In addition to the root-greening phenotype, PGA37 overexpression significantly hinders seedling growth and promotes somatic embryogenesis independently of cytokinin [21]. These phenotypes may also result from the activation of the cytokinin signaling pathway by PGA37 through the upregulation of ARR13 and ARR21, as the double mutation of ARR13 and ARR21 can nearly completely suppress the growth retardation phenotype caused by PGA37 overexpression (Figure 6A).

The interplay of the transcriptional regulators characterized in this study reinforces the importance of cross-family transcription factor regulatory networks in gene regulation. The PGA37-regulated module includes transcription factors from different families, enabling the coordination of complex gene expression programs. Such cross-family modules allow for the integration of multiple signaling pathways, enabling cells to respond to a wide range of internal and external stimuli (e.g., hormones and light). The interaction between transcription factors from different families provides combinatorial control over gene expression. These networks often exhibit redundancy, where different transcription factors can compensate for each other’s function. While some transcription factors have broad regulatory roles, others are more specialized. During development, cross-family networks are essential for cellular differentiation and tissue-specific gene expression. Cross-family transcription factor regulatory networks are essential for the dynamic, precise, and context-dependent regulation of gene expression, enabling organisms to adapt to their environment, develop properly, and maintain homeostasis. The PGA37-mediated regulatory module integrates the phytochrome and cytokinin signals to fine-tune the expression of genes involved in chloroplast development and chlorophyll biosynthesis in various tissues (Figure 7).

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The origins of pga37, myb118 (Salk_118812), myb115 (Salk_044168), and myb115 myb118 mutants, as well as the pER10-PGA37 transgenic line, have been previously described [21]. The arr13 (SALK_042719c) and arr21 (SALK_005772c) mutants, both in the Col-0 background, were generously provided by Professor Chen Shouyi from the Institute of Genetics and Developmental Biology. The glk2 (Salk_17006C) mutant and the GLK2 overexpression line (GLK2ox, CS9906) were acquired from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH). The 35S::PGA37-GR glk2 was generated by crossing the 35S::PGA37-GR overexpression line and the glk2 mutants. To create the arr13 arr21 double mutant, arr13 and arr21 were crossed, and this double mutant was subsequently crossed with 35S::PGA37-GR to produce 35S::PGA37-GR arr13, 35S::PGA37-GR arr21, and 35S::PGA37-GR arr13 arr21 mutants. PCR-based genotyping was employed to identify homozygous lines, with the primer sequences listed in Table S2.

For the light-independent analysis of chloroplast induction, seven-day-old seedlings grown on solid MS medium were transferred to liquid MS medium and cultured for an additional two days. The roots were then excised and cultured for seven days on MS medium, either supplemented with 10 μM 17 β-estradiol (Sigma-Aldrich, St. Louis,MO, USA, Cat# E8875) or without it, under both normal light and dark conditions.

Unless otherwise specified, Arabidopsis plants were grown at 22 °C under a 16 h photoperiod in soil or on agar plates containing half-strength Murashige and Skoog basal medium with vitamins (PhytoTechnology Laboratories^TM^, St Lenexa, KS, USA, Cat# M519), supplemented with 2% sucrose and 0.8% agar.

4.2. Plasmid Constructs and in Planta Transformation of Arabidopsis

To make the 35S:PGA37-GR fusion construct, a PCR fragment encoding the glucocorticoid receptor domain (amino acids 519 to 795) [23,32] was firstly amplified using primers GRF and GRB and introduced into the Sma I-Spe I sites of the HA-pBA plasmid. The resulting construct was designated HA-GR-pBA. Then, the PGA37 coding sequence without stop codon was inserted into the HA-GR-pBA vector using the Asc I and Spe I sites to generate the 35S:PGA37-GR construct. The primers used for plasmid construction are listed in Table S2. All constructs were thoroughly verified through extensive restriction digests and DNA sequencing analysis.

Arabidopsis transformation was performed using the floral-dip method [33] with the Agrobacterium GV3101 strain. T1 transgenic plants were selected on half-strength MS solid medium with kanamycin or Basta. Homozygous transgenic plants were obtained through self-crossing and subsequently used for analysis.

4.3. Chlorophyll Autofluorescence Detection and Ultrastructure Analysis of Root Plastids

To detect chlorophyll autofluorescence, the primary roots of two-week-old seedlings were examined approximately 1.5 cm from the root–hypocotyl junction using a laser confocal microscope (Leica, TCS SP8, Wetzlar, Germany). Chlorophyll autofluorescence was detected between 660 and 700 nm under 488 nm laser excitation and was merged with differential interference contrast images. For transmission electron microscopy analysis, the primary roots of two-week-old seedlings were cut approximately 1.5 cm from the root–hypocotyl junction and quickly prefixed overnight in 2.5% glutaraldehyde at 4 °C. The samples were then washed and postfixed in 1% OsO_4_ for about 4 h. After dehydration through a graded ethanol series (30%, 50%, 70%, 90%, and 100%, three times each, v/v), the samples were embedded in Epon812 resin. Ultra-thin sections, sliced to a thickness of 70 nm, were subsequently stained with 2% (w/v) uranyl acetate and lead citrate. Finally, the sections were photographed using a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan).

4.4. Chlorophyll Determination

To determine root chlorophyll content, 50 mg root samples were ground in 500 μL of 80% (v/v) acetone using a tissue lyser (Scientz-48, Scientz, Ningbo, China). The mixture was then centrifuged for 5 min at 10,000 g. The resulting supernatant was transferred to a new microtube, and the remaining precipitate was re-extracted with an additional 500 μL of 80% (v/v) acetone. The absorbance of the combined supernatants was measured at 646 and 663 nm using a BioSpectrometer^®^ kinetic spectrophotometer (Eppendorf). The chlorophyll (a and b) concentrations were calculated as described by Wellburn [34].

For the determination of seed chlorophyll content, fifty seeds at the torpedo stage were homogenized in 100 μL of dimethylsulfoxide (DMSO). The resulting extracts were then centrifuged for 5 min at 10,000× g. The supernatant was subsequently used to assess chlorophyll content. To identify the developmental stage, seeds from the middle of the silique were examined using a BX51 microscope (Olympus, Tokyo, Japan), while the other seeds within the siliques were utilized for chlorophyll measurement.

Chlorophyll assessments were conducted in three independent experiments for embryos and four independent experiments for roots.

4.5. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR

Total RNA was extracted using the RNAprep pure Plant Kit with DNase I (Tiangen Biotech (Beijing), China, Cat# DP432). First-strand cDNA synthesis was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). The resulting cDNA was diluted 10- to 100-fold, and quantitative transcript analysis was conducted on a Bio-Rad CFX96 system using SYBR^®^ Fast qPCR Mix (Takara Biotechnology, Dalian, China, Cat# RR430). All procedures were carried out according to the manufacturer’s instructions.

4.6. Dexamethasone Treatment, Library Construction, RNA-Seq and Differential Expressed Gene Analysis

Two-week-old 35S:PGA37-GR seedlings, geminated and grown on solid MS medium, were transferred to liquid MS medium containing 10 μM DEX (Sigma, Cat# D4902) and 100 μM CHX (MedChemExpress, Cat# HY-12320). Seedlings treated with 100 μM CHX were used as the control. After 4 h of treatment, the seedlings were collected and then total RNA was extracted, as described above. Library construction, RNA-Seq, and differentially expressed gene analysis were performed as described previously [35].

4.7. Protein Expression of PGA37 and Biolayer Interferometry Assay

To express the PGA37 protein, the coding sequence (CDS) of PGA37 was cloned into the NdeI and EcoRI restriction sites of the pET-28a (+) vector, allowing for the expression of PGA37 with an N-terminal histidine tag for affinity purification and immunoblotting. The constructed pET28-PGA37 plasmid was then transformed into BL21 (DE3) competent cells. The transformed cells were cultured induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 37 °C. After incubation, the cells were harvested, and the expressed protein was purified using standard purification techniques.

The purified PGA37 protein was then subjected to a biolayer interferometry (BLI) assay to evaluate its binding interactions with various oligonucleotides using a biolayer interferometry system (BLItz, FortèBIO Inc., Fremont, CA, USA). 5′-Biotinylated DNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), with the sequences detailed in Table S2. The biotinylated oligonucleotides were immobilized onto streptavidin biosensor tips that had been pre-wetted for 10 min in kinetic buffer containing PBS, 0.1% BSA, 0.02% Tween-20, and 0.05% sodium azide. The assay steps were initial baseline (60 s), loading (300 s), baseline stabilization (240 s), ligand–analyte association (300 s), and ligand–analyte dissociation (300 s).

5. Conclusions

In summary, our findings highlight the multifaceted role of PGA37 in regulating chloroplast development in Arabidopsis. We demonstrated that ectopic expression of PGA37 induces chloroplast formation in roots through GLK2 and cytokinin signaling mediated by ARR13 and ARR21, providing new insights into the intricate regulatory networks that govern plant developmental processes. Future studies should focus on delineating the specific roles of ARR13 and ARR21 in chloroplast development and investigating potential downstream targets that mediate the effects of PGA37 beyond its currently known functions. Additionally, exploring the potential of PGA37 for manipulating this pathway in agricultural crops could offer valuable strategies for enhancing growth and yield under varying environmental conditions.