Heavy Metal-Associated Isoprenylated Plant Proteins (HIPPs): Multifunctional Regulators of Metal Homeostasis, Abiotic/Biotic Stress Responses, and Plant Development

1 Introduction

Heavy metal pollution, abiotic stresses (such as cold, drought, and salinity), and pathogen infections are major environmental constraints that severely threaten global crop production and food security [1,2]. Among these, the non-essential heavy metal cadmium (Cd) raises particular concern owing to its high toxicity, persistence in soils, and ease of uptake by crop plants. Excessive Cd not only inhibits plant growth, reduces photosynthesis and biomass accumulation, but also enters the food chain, causing serious health problems, including cancer, renal dysfunction, and osteoporosis in humans [3,4]. Copper (Cu), an essential micronutrient, becomes toxic when present in excess, leading to oxidative stress and impaired growth [5]. Similarly, biotic stresses such as bacterial, fungal, and nematode infections cause devastating yield losses worldwide [6,7]. To cope with these diverse environmental challenges, plants have evolved intricate regulatory networks involving metal chaperones, transporters, and signaling molecules that maintain ion homeostasis and trigger appropriate defence responses [8,9].

Key players in metal homeostasis and stress signaling include HIPPs [10,11,12,13]. HIPPs are a plant-specific subfamily of metallochaperones, characterized by the presence of one or two HMA domains at their N-terminus and a C-terminal isoprenylation motif (CaaX box) [14,15]. HIPPs are strictly defined by the simultaneous presence of at least one HMA domain and a C-terminal CaaX isoprenylation motif. In contrast, HPPs (heavy metal-associated plant proteins) that possess only HMA domains without a CaaX motif are not classified as HIPPs and are discussed only when functionally linked to HIPPs. The HMA domain contains a conserved Cys-XX-Cys motif that directly binds transition metal ions such as Cd2+, Cu2+, Zn2+, and Pb2+, enabling metal chelation and detoxification [16,17]. The C-terminal isoprenylation motif undergoes post-translational prenylation, which anchors the protein to the plasma membrane or other endomembranes and facilitates protein-protein interactions essential for signal transduction [18,19]. This unique dual-domain architecture distinguishes HIPPs from other metallochaperones that possess only HMA domains. Moreover, HIPP genes are found exclusively in vascular plants, suggesting that they emerged as an evolutionary adaptation to the complex terrestrial environment.

This review critically synthesizes the HIPP literature, distinguishing between mechanistically validated functions and correlative expression data. We prioritize studies that provide functional validation (e.g., mutant or overexpression lines, protein–protein interactions, in vitro binding assays) and highlight contradictory findings as well as knowledge gaps. A systematic literature search of PubMed, Web of Science, and Google Scholar was conducted for the period January 2000–March 2026 using the keywords “HIPP”, “heavy metal-associated isoprenylated plant protein”, “metallochaperone plant”, “HMA domain isoprenylation”, and “farnesylated plant protein”. Compared with earlier reviews (e.g., de Abreu Neto et al., 2013; Barr et al., 2023), this work integrates the rapid expansion of knowledge from over ten crop species published in 2024–2026, provides a mechanistic classification of HIPP in plant metal homeostasis, abiotic/biotic stress responses, and plant development, and offers a translational roadmap for agriculture.

Initial studies on HIPPs focused primarily on their roles in heavy metal detoxification and homeostasis. In Arabidopsis thaliana (Arabidopsis), AtHIPP20, AtHIPP22, AtHIPP26, and AtHIPP27 complement Cd-sensitive yeast mutants, whereas the athipp20/21/22 triple mutant displays increased Cd sensitivity and reduced Cd accumulation [18,20]. In rice, OsHIPP29 is upregulated by Cd and Zn; Overexpression of OsHIPP29 reduces Cd accumulation, thereby improving growth under Cd stress, while knockout lines are more susceptible [21]. OsHIPP16 and OsHIPP56 have also been characterized as positive regulators of Cd tolerance and suppressors of Cd accumulation in grains [22,23]. These studies firmly established HIPPs as central regulators of metal stress responses.

In recent years, the functional scope of HIPPs has expanded dramatically. They are now known to participate in abiotic stress responses beyond heavy metals. Recently, a genome-wide identification in tobacco (Nicotiana tabacum) and analysis of stress-responsive expression patterns provided a foundation for understanding HIPP functions [24]. A pan-genome analysis and expression profiling of the HIPP gene family in cassava revealed that a total of 59 MeHIPP pan-genes were identified and classified into core, softcore, dispensable, and private categories, with uneven chromosomal distribution. The gene family expanded mainly through segmental duplication, with most members under purifying selection. Expression profiles indicated roles in response to Xanthomonas phaseoli pv. Manihotis (Xpm) infection, cadmium and drought stress, with varying tissue- and cell-specific transcript levels [25]. For example, barley HvFP1, a member of the barley HIPP gene family, is induced by drought, cold, and ABA, and overexpression of HvFP1 delays leaf senescence [26]. In Arabidopsis, AtHIPP26 interacts with the zinc finger homeodomain transcription factor ATHB29 under cold, salt, and drought stress [27], but the exact mode of action of the two regulatory factors HIPP26 and ATHB29 in response of the plants to different stress conditions has to be solved in future experiments. In contrast, grapevine VvHIPP21 acts as a negative regulator of cold and drought tolerance [28]. VvHIPP21 expression is suppressed by cold and ABA but induced by drought, and the protein localizes to both the cytoplasm and nucleus. Ectopic expression of VvHIPP21 in Arabidopsis alleviates heavy metal (Cu2+, Cd2+) toxicity and reduces drought tolerance [28]. Regarding biotic stress, HIPP proteins are emerging as common targets of pathogen effectors. The rice blast fungus Magnaporthe oryzae effector AVR-Pik directly binds to the HMA domain of rice OsHIPP19 to suppress immunity [29]. The kiwifruit bacterial canker pathogen Pseudomonas syringae pv. actinidiae (Psa) effector AvrPto5 targets AcHIPP26 directly [30], but the function of AcHIPP26 in Psa resistance is not clear. The movement protein TGB1 of potato mop-top virus interacts with NbHIPP26 to facilitate the virus's long-distance movement [31], and the beet cyst nematode Heterodera schachtii relies on AtHIPP27 to complete successful parasitism [32]. These findings indicate that HIPP proteins play an important role in pathogen infection processes. Furthermore, HIPPs also regulate plant development and hormone signaling. HIPP influences the stability of cytokinin oxidase (CKX) by regulating the endoplasmic reticulum-associated degradation (ERAD) pathway in Arabidopsis, thereby finely modulating cytokinin signaling and plant development [33].

Despite these significant advances, several important questions remain unresolved. For example, The molecular mechanisms that how exactly metal ions are transferred from HIPPs to downstream transporters, and how isoprenylation dynamically control HIPP subcellular localization and function are not clear. Additionally, although genome-wide identification of HIPP genes has been performed in several crops, their agricultural potential remains largely unexplored. Notably, the role of HIPPs as pathogen effector hubs has only recently been recognized, whereas the interplay between HIPP-mediated metal homeostasis and plant immunity requires further investigation.

In this review, we aim to introduce the structural features and multifaceted functions of HIPP proteins in heavy metal detoxification, abiotic and biotic stress responses, hormone signaling and plant development. We also discuss the molecular mechanisms underling these related functions. We highlight the potential applications of HIPP genes in stress tolerant and low cadmium crop breeding and outline future research directions to address current knowledge gaps.

2 Structural Characterization of Plant HIPPs

HIPPs are metallochaperones that contain conserved structures, including 1–2 heavy metal-associated domains (HMAs), a C-terminal isoprenylation motif, as well as a flexible glycine-rich and proline-rich region located between these two domains [27]. Here, we generated six representative HIPP proteins (OsHIPP5, OsHIPP9, OsHIPP19, OsHIPP28, OsHIPP42, and OsHIPP56) from rice for protein structure analysis by using AlphaFold 3. We provided UniProt accession numbers and domain boundaries (HMA and CaaX positions) for each protein (Fig. 1), as well as the pLDDT scores, and predicted aligned error (PAE) plots (Fig. 2). Structural analysis of the six predicted structures confirmed that HMA domain cores are conserved while loop regions and C-terminal extensions diverge. For example, OsHIPP56 lacks a CaaX motif at its C-terminus, indicating that it may belong to the HPP gene family, whereas OsHIPP28 contains two conserved HMA domains based on the prediction results. These findings confirm strong conservation of the metal-binding fold between HIPP and HPP proteins. However, differences in the CaaX isoprenylation motif suggest that these HIPP or HPP proteins may interact with distinct protein partners, although direct functional divergence awaits experimental validation (Fig. 1). A genome-wide identification of heavy metal-associated proteins across angiosperms and ancestral plants has revealed evolutionary conservation and functional insights into HIPP proteins [34].

The HMA domain is defined by a ferredoxin-like structural fold (βαββαβ), spans approximately 70 amino acids, and harbors the Cys-XX-Cys metal-binding motif within its first loop, as confirmed by the crystal structure of the OsHIPP19 [8,14,27]. In the yeast Antioxidant Protein 1 (ATX1) protein, the coordination of cysteine residues in the HMA domain with Cu2+ is critical for the transfer of Cu2+ between ATX1 and copper transporters [35]. For example, ATX1 can interact with Ca2+-sensitive Cross Complementer (CCC2), a Golgi membrane-localized, Cu-transporting P-type ATPase, at the site where copper is loaded into newly synthesized metalloproteins [8,36]. Isoprenylation (also called prenylation) is a post-translational modification in which a 15-carbon farnesyl group or 20-carbon geranylgeranyl group is covalently attached via a thioether bond to the cysteine residue of a C-terminal CaaX motif, where 'C' represents cysteine, 'a' represents an aliphatic amino acid, and 'X' represents any amino acid [18,37]. Recent studies have found that poplar HIPPs are enriched in plasmodesmata and they may regulate intercellular metal ion or signaling molecule transport through lipid modification [12]. In addition, isoprenylation is also involved in dynamic subcellular localization transitions in response to stress. For example, the rice OsHIPP41 migrates from cytoplasm to perinuclear region under cold stress and activates the expression of stress-resistant genes [10]. More recent research has demonstrated that prenylation is largely required for the localization of AtHIPP7 to plasmodesmata in Arabidopsis [33]. However, the molecular mechanism by which isoprenylation dynamically controls HIPP subcellular localization and function remains unclear. Moreover, prenylated proteins are also important regulators of signal transduction, cytoskeletal organization and intracellular vesicle transport [37].

Figure 1: Predictions of HIPP proteins’ structure in rice plants using AlphaFold 3. (A–F) Six representative rice HIPP proteins involved in metal homeostasis and plant immunity were analyzed for protein structure. The HMA domain and the isoprenylation motif positions and sequences in OsHIPP42, OsHIPP9, OsHIPP19, OsHIPP28, OsHIPP56, and OsHIPP5 are highlighted in a black box. UniProt accession numbers are provided in parentheses.

Figure 2: LDDT and PAE of predicted OsHIPPs proteins obtained using AlphaFold 3. LDDT: a per-residue confidence score that ranges from 0 to 1. Higher values (typically >0.9) indicate high confidence in the placement of local atoms, suggesting that the positions of the backbone and side chains are accurate (left panels). PAE: a heatmap that shows the confidence level of the model's prediction for the relative positions of residue pairs. Low PAE values indicate well-defined spatial relationships, while high values suggest uncertainty in the positioning of domains or chains. LDDT and PAE of predicted OsHIPP42 (A,B); OsHIPP9 (C,D); OsHIPP19 (E,F); OsHIPP28 (G,H); OsHIPP56 (I,J); and OsHIPP5 (K,L).

3 HIPPs Participate in Homeostasis and Detoxification of Heavy Metal Ions

3.1 Direct Metal Binding and Biochemical Evidence

Many studies have shown that plant HIPP proteins bind directly to metal ions (Table 1). For example, metal-chelate affinity chromatography experiments showed that recombinant ATFP3 (AtHIPP07) could bind Cu2+, Ni2+ and Zn2+ while AtFP6 (AtHIPP26) could also bind Cu2+ Pb2+, and Cd2+ in vitro [14,38]; Similarly, AtCdI19 (AtHIPP06) protein was detected by circular dichroism (CD) spectroscopy and shown to bind Cu2+ and Hg2+ [19]; Furthermore, purified AtHIPP33 protein directly bound with Cd in vitro [39]. Using co-incubation of proteins with heavy metal salts, Chen and Xiong [40] demonstrated that OsHIPP24 can chelate Cd2+ and Cu2+ in vitro. These assays establish direct metal-binding capacity, though the metal-binding stoichiometry and affinity constants remain largely undetermined for most HIPPs.

3.2 Protein-Protein Interactions in Metal Transfer

It has been reported that HIPPs may interact with other factors to facilitate the exchange of metal ions, ensuring proper transport and distribution of metals and maintaining metal homeostasis in plants [41]. Take Saccharomyces cerevisae ATX1 protein as an example: the MXCXXC motif in ATX1 interacts with the Cu2+-binding domain (MTCXXC) of CCC2 indicating that ATX1 donates Cu2+ to CCC2 [42]. In Arabidopsis, the heavy-metal-binding farnesylated protein AtFP6 (AtHIPP26) interacts with acyl-CoA-binding protein ACBP2 to confer tolerance to Cd. Since both AtFP6 and ACBP2 bind Pb2+, Cd2+, and Cu2+, Plasma membrane-localized ACBP2 and AtFP6 likely mediate Pb2+, Cd2+ and Cu2+ transport in Arabidopsis roots [38]. Moreover, the R2R3-MYB transcription factor MYB49 binds to the promoter of AtHIPP22 and AtHIPP44, resulting in upregulation of their expression and subsequent Cd accumulation in Arabidopsis [43]. In apple (Malus domestica), MdWRKY17 acts upstream of MdHIPP1 to enhance its expression and increase Cd tolerance by reducing Cd accumulation [44]. In Arabidopsis, the core autophagy protein ATG8e associates with HIPP33 and recruits it for autophagic degradation in an AIM (ATG8-interacting motif) dependent manner [39]. AtHIPP33 acts as a cargo receptor to modulate the Cd response by facilitating autophagy-mediated vacuolar sequestration of Cd. However, how ATG8 recruits AtHIPP33 remains unknown. Indeed, the exact mechanism by which metal ions are transferred from HIPPs to downstream transporters is still unresolved.

3.3 Transcriptional Responses and Functional Validation in Planta

HIPP genes respond to a variety of heavy metal stresses. The expression of AtHIPP06 (AtCdI19) was induced by Cd2+, Hg2+, Fe2+, and Cu2+, while the expression of AtHIPP26 (AtFP6) was induced by Cd2+ and Zn2+ in Arabidopsis [19,38]. The expression of OsHIPP16, OsHIPP24, and OsHIPP42 was upregulated by Cu2+ and Cd2+, whereas the expression of OsHIPP33 was induced by Zn2+ and Fe2+ in rice [45,46]. Cd stress led to upregulation of SlHIPP7, SlHIPP21, SlHIPP26, and SlHIPP32 in tomato roots [47]. The expression of HIPP26 and HIPP27 in Brassica oleracea var. Acephala (kale) was upregulated by CdCl2 [13]. In addition, the expression level of OsHIPP28 substantially increased in shoots but decreased in roots compared with the control under Mn or Cd treatment, whereas expression of OsHIPP34 in roots was significantly induced under Mn, Cd, and Cu stress, but in shoots it was significantly reduced only under Mn stress [17]. In maize, Cd treatment confirmed that the expression levels of ZmHIPP11, ZmHIPP30, and ZmHIPP48 were generally higher in shoots than roots, while ZmHIPP02 and ZmHIPP57 exhibited the opposite pattern [48]. In soybean (Glycine max), six genes (GmHIPP9, GmHIPP13, GmHIPP29, GmHIPP43, GmHIPP58, and GmHIPP73) were highly induced by Al [49]. These results indicate that the expression levels of HIPP family members differ across plant tissues under heavy metal stress, suggesting that HIPP family members may play distinct roles in metal ion homeostasis and detoxification. It is important to note that transcriptional responsiveness (Class III evidence) alone does not demonstrate a functional role. We highlight cases where this has been validated by mutant or overexpression studies below.

Through genetic identification, functional characterization of HIPPs in heavy metal homeostasis has been well studied. Heterologous expression of AtHIPP20, AtHIPP22, AtHIPP26, and AtHIPP27, as well as OsHIPP09 and OsHIPP16, in yeast enhanced the tolerance of cadmium-sensitive ycf1 yeast mutant to cadmium, suggesting that HIPP may have Cd-detoxifying effects [17,18,20]. Moreover, heterologous expression of OsHIPP17 in copper-sensitive yeast mutants decreased the tolerance to copper [50]. Similarly, heterologous expression of tea plants CsHIPP22, CsHIPP24, and CsHIPP36 in Cd-sensitive ycf1 yeast mutant improved tolerance to cadmium, with CsHIPP24 conferring much greater cadmium tolerance than CsHIPP22 and CsHIPP36 [51]. The HIPP gene family in Nelumbo nucifera G. (lotus) plays a crucial role in Cd resistance, as NnHIPP14, NnHIPP21, and NnHIPP33 conferred varying degrees of Cd tolerance when overexpressed in yeast [52]. Functional validation in yeast demonstrated that sorghum SbHIPP40 overexpression enhanced Cd tolerance in the ycf1 mutant strain [53]. Heterologous expression of Solanum americanum SaHIPP41 in yeast enhanced Cd accumulation and sensitivity, a function dependent on its conserved CxxC domain [54]. Overexpression of TaHIPP1 in the yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu2+ stresses [55]. Collectively, these findings suggest that while certain HIPP members are conserved in Cd accumulation, the family as a whole exhibits functional plasticity across different metals and physiological outcomes.

Recent studies have also examined the function of HIPPs in Cd accumulation in relevant transgenic plants. For example, overexpression of AtHIPP06 and AtHIPP26 confers tolerance to Cd, while mutant athipp20/21/22 increases sensitivity to cadmium in Arabidopsis [18,19]. The growth of OsHIPP16/29/56 overexpression rice plants improved significantly under excessive Cd treatment [21,22,23]. More recently, overexpression of IbHIPP7 in sweet potato reduced Cd accumulation and alleviated Cd toxicity, suggesting its potential for phytoremediation or safe crop production [56]. The metallochaperone OsHIPP53 reduces Cd accumulation specifically in rice roots, likely by sequestering Cd and limiting its translocation to shoots [57]. In Medicago sativa (alfalfa), overexpression of MsHIPP12 in Arabidopsis enhances cadmium tolerance by increasing antioxidant enzyme activity and promotes cadmium detoxification by reducing Cd uptake in transgenic lines and mitigating oxidative stress [58]. SbHIPP40 overexpression rice plants accumulated 1.68- to 3.92-fold higher Cd in stems, leaves, and grains compared with wild-type plants, indicating its potential functional role in Cd tolerance in sorghum [53]. The regulatory mechanisms of macroautophagy/autophagy in plant tolerance to Cd remain poorly elucidated. A Recent study showed that overexpression of SaHIPP41 in the hyperaccumulator Solanum americanum enhances Cd accumulation and improves tolerance to Cd stress, offering a valuable gene for phytoremediation [54]. Although HIPPs are involved in Cd accumulation, a striking pattern emerges: some HIPPs reduce Cd accumulation in shoots and grains (e.g., OsHIPP29, OsHIPP53, IbHIPP7), while others promote Cd accumulation, especially in hyperaccumulator species (e.g., SaHIPP41). We propose that HIPPs involved in vacuolar sequestration or root retention reduce shoot/grain Cd, whereas those that enhance xylem loading or leaf storage increase whole-plant Cd accumulation. This distinction is critical for breeding low-Cd crops versus engineering plants for phytoremediation.

In addition to Cd, HIPPs are also involved in other metal metabolism processes in plant cells. For example, GmHIPP29 overexpression significantly increased Al accumulation in the cell sap of the transgenic soybean hairy root tips, leading to increased Al sensitivity [49]. However, other GmHIPP genes, such as GmHIPP9, GmHIPP13, GmHIPP43, GmHIPP58, and GmHIPP73, are also significantly induced by Al. Whether these homologous genes have similar or redundant functions requires further investigation. A high-mobility group protein, MdHMGB15, enhances zinc (Zn) tolerance in apple by activating multiple metal homeostasis genes, including MdHIPP37, thereby improving metal detoxification and reducing Zn-induced oxidative damage [59]. Notably, research on HIPPs in metal metabolism (other than Cd) is still limited, which is of great interest to the global scientific community.

In addition to binding heavy metal ions or interacting with other proteins to regulate the metal homeostasis in plants, HIPPs can also regulate hormone-dependent signaling pathways to maintain metal homeostasis. Melatonin mediates lead (Pb) accumulation reduction in radish taproot via the RsWRKY26-RsHIPP26-RsASMT2 module, integrating melatonin signaling with HIPP-mediated metal detoxification [60]. In tomato, SlHIPP4, SlHIPP7, SlHIPP9, SlHIPP21, SlHIPP26, and SlHIPP32 under cadmium stress caused a decrease in root length and plant height, as well as a decrease in the levels of ABA and salicylic acid (SA), while SlHIPP7 and SlHIPP32 led to a decrease in Methyl jasmonate (MeJA) levels [45]. These results suggest that HIPP may respond to metal stress through ABA-, SA-, and MeJA-dependent signaling pathways, and the underlying mechanism should be further investigated.

4 HIPPs Are Involved in the Response to Abiotic Stress in Plants

Besides chelating cytoplasmic metal ions to mediate heavy metal accumulation and detoxification, HIPPs may also function in other abiotic stress responses, as evidenced by the responsiveness of several rice HIPP genes to cold, drought, and salt stress [10] (Table 2). We distinguish between transcriptional responsiveness (Class III) and functionally validated roles (Class II) below. For example, OsHIPP23 and OsHIPP15 are downregulated under drought and salt stress, respectively. OsHIPP41 exhibits upregulation under both drought and cold conditions. In contrast, OsHIPP11 and OsHIPP45 show downregulation exclusively in response to cold stress. Although expression profiling suggests involvement, functional proof via mutant analysis is lacking for OsHIPP11 and OsHIPP45 in cold stress. Genome-wide identification of HIPP genes and subsequent functional analysis of GsHIPP79 in wild soybean demonstrated that GsHIPP79 is involved in the alkaline stress response, expanding the knowledge of HIPPs in response to abiotic stress beyond heavy metals [68].

4.1 Protein–Protein Interaction-Mediated Stress Regulation

HIPPs regulate abiotic stress tolerance by interacting with other proteins. In Arabidopsis, AtHIPP26 is induced during drought, cold, and salt stress. ATHB29, a zinc-finger homeodomain transcription factor involved in drought stress response, was shown to interact strongly with AtHIPP26 through the two central cysteines of the AtHIPP26 heavy metal-associated domain [27]. However, the interaction between HIPP26 and ATHB29 in response to different stress conditions requires further investigation. In Chenopodium quinoa, CqHIPP34 can interact with a drought-responsive zinc finger homologous domain protein CqZF-HD14. Transient overexpression of ZF-HD14 or CqHIPP34 alone enhances drought tolerance, while transient co-overexpression of CqHIPP34 and CqZF-HD14 results in a significant increase in drought tolerance. This result suggests that they may cooperate to withstand drought stress [69]. In sweet cherry, a basic helix-loop-helix protein, PavHLH106, interacts with PavHIPP16 to positively regulate the cold tolerance of tobacco [70,71]. Interestingly, in grape, VvHIPP21 negatively regulates the cold tolerance by interacting with the E3 ubiquitin ligase High Expression of Osmotically Responsive Gene 1, VvHOS1, to co-regulate cold stress [28]. These opposite functions may arise from species-specific evolutionary adaptations, differences in interacting partners, or distinct downstream signaling pathways. Therefore, the role of HIPP proteins in cold response is not inherently conserved but rather depends on the broader regulatory context within each species. Further comparative studies are needed to elucidate the molecular mechanisms underlying such functional diversification. The HIPP26L-Nuclear Factor Y subunit C9 (NF-YC9)-Salt Responsive MYB Transcription Factor (SRMT) module regulates drought response, demonstrating a signaling pathway where an HIPP protein participates in abiotic stress adaptation in poplar [72]. It is noteworthy that the experiments were restricted to poplar seedling saplings in a greenhouse. Because poplar is a perennial species, future studies should extend the growth period and incorporate field experiments to achieve a more comprehensive perspective.

4.2 Hormone Pathway Associations

In addition to protein interactions, HIPP genes also play a role in the phytohormone signaling networks that operate under abiotic stress. Although HvFP1 is induced by drought, senescence, and ABA treatment, its overexpression represses ABA biosynthesis-related genes, suggesting that HvFP1 participates in a negative feedback loop within the ABA signaling pathway [26]. In tomato, SlHIPP4, SlHIPP7, SlHIPP9, SlHIPP21, SlHIPP26, and SlHIPP32 physically interact with hormone-responsive transcription factors in yeast two-hybrid assays, and their co-expression with ABA- and JA-signaling components suggests they form stress-responsive protein complexes [47]. However, observed changes in hormone levels should be viewed as possible signaling associations rather than confirmed causal mechanisms unless supported by genetic epistasis or inhibitor studies.

5 HIPPs Are Involved in Plant-Pathogen Interactions

Plants can recognize effector proteins secreted by microbial plant pathogens by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, which contain an HMA domain or other proteins like some HIPPs to induce an immune response [45,77] (Table 3). HIPP roles in immunity can be classified as effector targets, susceptibility/resistance factors, or components of ubiquitination/receptor-associated immune pathways.

5.1 HIPPs as Effector Targets

AVR-Pik is an effector protein secreted by Magnaporthe oryzae during infection of rice leaves, and it exists in multiple variants. To date, six AVR-Pik (A-F) variants have been identified in rice [29]. The study demonstrates that rice OsHIPP19 is a genuine virulence target of the Magnaporthe oryzae effector AVR-Pik. The effector binds the HMA domain of OsHIPP19 with high affinity, including stealthy variants such as AVR-PikC and AVR-PikF that escape nucleotide-binding and leucine-rich repeat (NLR) domain-containing immune receptor Pik-1. In contrast, the integrated OsHIPP19-HMA domain of the NLR receptor Pik-1 acts as a decoy that structurally mimics the HIPP target and directly binds AVR-Pik to trigger immunity. This highlights the pathogen’s evolutionary strategy to bypass host immunity while retaining its ability to interact with host targets critical for virulence [77]. Notably, OsHIPP19 is not a helper component, nor is it physically integrated into an NLR receptor; instead, it is a host susceptibility factor that the pathogen manipulates, and scientists have independently co-opted its HMA domain into an NLR as a surveillance module. The Magnaporthe oryzae effector Pwl2 alters HIPP43 localization in host cells to suppress plant immunity, demonstrating a pathogen strategy to manipulate host metal-associated proteins [78]. Moreover, a recent study found that a novel effector, MgMO289, interacts with a new rice copper metallochaperone OsHPP04. Overexpressing OsHPP04 or MgMO289 exhibited an increased susceptibility to Meloidogyne graminicola [79]. OsHIPP28 contains two HMA domains and is preferentially expressed in leaf blades and sheaths, implicating it in defense mechanisms associated with photosynthetic tissues. The interaction between OsHIPP28 and the fungal effector protein RsMf8HN has been experimentally validated. This interaction likely facilitates pathogen infection by interfering with host metal metabolism or immune signaling pathways, thereby attenuating plant defense responses [80]; however, this mechanism requires further investigation.

5.2 HIPPs as Susceptibility or Resistance Factors and Components of Ubiquitination/Receptor-Associated Immune Pathways

Isopreneylated motifs and proline-rich regions in HIPPs also play an important role in pathogen resistance [75]. OsHIPP5, also known as Pi21, is a negative regulator in rice blast disease. Pi21 may help optimize defense mechanisms by slowing the initiation of plant defense responses, but it weakens resistance to the pathogen Magnaporthe oryzae in both rice and Arabidopsis [81,82]. The pi21 mutant is missing a proline-rich region. pi21-mediated resistance does not rely on hypersensitive response (HR), but is achieved by delaying the expansion of pathogenic hyphae between cells [81]. These findings suggest that the integrity of the proline-rich motif in the OsHIPP5 protein is closely associated with susceptibility to rice blast disease. Additionally, the role of ubiquitination in HIPP-mediated immunity is also of great interest. HIPP1-V from Haynaldia villosa positively regulates resistance to powdery mildew (Pm) caused by Blumeria graminis f. sp. tritici (Bgt). Overexpression of HIPP1-V significantly inhibited haustorium formation, reduced the infection index, and provided broad-spectrum resistance against 10 Pm isolates. Importantly, isoprenylation of HIPP1-V was essential for its plasma membrane (PM) localization, interaction with the E3 ligase CMPG1-V, and Pm resistance function [75]. The prenylation modification ensures that HIPP1-V localizes to the plasma membrane and recruits CMPG1-V to the PM to form a defense complex. Inhibition of isoprenylation, either by mutating the CaaX motif or using the inhibitor tipifarnib, results in the shedding of HIPP1-V from the PM and significantly reduces disease resistance [75]. OsFBX388, an E3 ligase, orchestrates the ubiquitination of OsHIPP56 and blast fungus MAX effectors in a coordinated manner, a process that regulates rice immunity [83]. Notably, the question remains unresolved whether the “dual ubiquitination” strategy, whereby OsFBX388 ubiquitinates both host HIPP proteins and pathogen effectors, commonly exists in other plant-pathogen interaction systems. How is this dynamic balance regulated, and what are the mechanisms underlying the conservation and specificity of ubiquitination-mediated regulation among different HIPP proteins?

5.3 The Metal–Immunity Interface

Moreover, HIPP can also be involved in the response of pathogenic microorganisms through hormone-dependent pathways. In Arabidopsis, the zinc-binding nuclear protein HIPP3 has been identified as a dual-function regulator that operates upstream of both the SA-dependent immune pathway and flowering time regulation. Zschiesche et al. [84]. demonstrated through experimental inoculation with the pathogenic bacterium Pseudomonas syringae pv. Tomato, that AtHIPP3 expression undergoes rapid and significant induction following pathogen infection in Arabidopsis leaves. Mechanistic studies further revealed that HIPP3 acts as a negative regulator of the SA signaling pathway. This regulatory function may be mediated through its zinc-binding capacity, potentially modulating the activity of key immune proteins such as Nonexpressor of pathogenesis-related genes 1, NPR1, thereby suppressing the activation of SA-dependent immune responses [84]. Chitin elicitor receptor kinase 1, OsCERK1, physically interacts with and phosphorylates OsHPP08, regulating copper uptake and conferring blast resistance in rice, linking copper homeostasis with disease resistance [61], which deepens our understanding of the intricate interplay between biotic and abiotic signals in rice. The VlMYB149-VlHIPP30 regulatory module confers enhanced resistance to Botrytis cinerea in grapevine by upregulating the antioxidant system and copper metabolism [66]. The emerging picture is that pathogens target HIPPs to perturb host metal pools, suppress reactive oxygen species (ROS) bursts, and subvert hormone signaling. Conversely, plants may have co-opted these metallochaperones as surveillance components. However, the exact molecular consequences for immunity remain unresolved for several interactions, and definitive evidence of immune signaling function requires further genetic dissection.

6 HIPP Is Involved in Plant Growth and Development

HIPP may also be involved in the regulation of plant growth and development (Table 3). For example, overexpression of PavHIPP16 in tobacco promotes germination and root elongation [76]; however, the underlying mechanism remains unknown. Additionally, overexpression of AtHIPP3 resulted in delayed flowering in Arabidopsis; however, the exact mode of action of AtHIPP3 in flower development has not been clarified [84]. Moreover, long-term field trials demonstrated that the mutation of OsHIPP33 resulted in shortened internodes, decreased tillering, stunted panicle development, and significantly reduced grain fertility and yield [46], but the underlying mechanism by which OsHIPP33 regulates agronomic traits requires further investigation.

6.1 Cytokinin Signaling and ERAD

The plant hormone cytokinin mediates diverse developmental processes, including differentiation in meristems and cell proliferation and developing organs, the onset of senescence, and responses to environmental signals [33]. HIPP affects the stability of cytokinin oxidase (CKX) by regulating the endoplasmic reticulum-associated degradation (ERAD) pathway in Arabidopsis, thereby finely regulating cytokinin signaling and plant development [33]. Specifically, AtHIPP7 interacts with CKX1 and promotes its degradation via the ERAD pathway, leading to elevated cytokinin levels. This results in altered root meristem size, delayed leaf senescence, and modified shoot architecture. The interaction is mediated by the isoprenylation motif of HIPPs, revealing a prenylation-dependent regulatory mechanism that directly impacts developmental programs. Nevertheless, the mechanism by which HIPPs execute their function remains unclear. It remains to be determined, for example, whether HIPPs act solely through interaction with ERAD substrates or whether they also target components of the ERAD machinery.

6.2 Direct vs. Indirect Developmental Effects

Notably, although the HIPP proteins have been demonstrated to be involved in the development of many species, the underlying molecular mechanism remains largely unknown. It is critical to distinguish whether developmental phenotypes are direct consequences of HIPP activity or secondary effects of altered metal homeostasis and hormone signaling. For CKX-regulating HIPPs, the effect on cytokinin signaling is likely direct [33]. For OsHIPP33, the dwarf and fertility phenotypes may arise from both Zn/Fe deficiency-induced metabolic stress and altered hormonal balance [46]. Future studies should uncouple these possibilities using tissue-specific complementation and metal supplementation experiments.

7 Conclusions and Future Perspectives

Plant HIPPs are a unique family of metallochaperones that integrate heavy metal homeostasis, abiotic stress tolerance, and pathogen resistance through their HMA domains and isoprenylation motifs (Fig. 3). Recent genome-wide studies across multiple crop species have greatly expanded our understanding of HIPP family diversity and stress-responsive expression patterns. Functional analyses have revealed both positive and negative regulators of metal tolerance, as well as novel mechanisms such as autophagy-mediated Cd sequestration and ubiquitination-based immune regulation. A conceptual model emerges in which HIPPs serve as molecular hubs that coordinate environmental sensing, metal buffering, and signaling across multiple stress and developmental pathways.

Future research should focus on a set of unresolved problems and potential applications of HIPPs in agriculture. For example, elucidating metal transfer mechanisms will require structural biology approaches such as cryo-EM, together with in vitro reconstitution assays, to determine how HIPPs deliver metal ions to downstream transporters. The dynamic regulation of isoprenylation can be investigated in parallel using chemical biology tools, including photoactivatable prenyl probes, combined with live-cell imaging to track HIPP relocalization in real time after exposure to stress. In order to overcome functional redundancy, which has long masked single-mutant phenotypes, the CRISPR-based multiplex editing of HIPP genes, as well as single-cell transcriptomics can help exploring their core functions. It is important to engineer HIPP specificity via structure-guided mutagenesis which offers the possibility of altering metal selectivity, for example, enhancing Cd binding for phytoremediation or designing HIPP decoys that interacting with pathogen effectors without disrupting endogenous metal homeostasis. On the other hand, low-cadmium crop varieties could be developed by overexpression or gain-of-function of HIPP genes under root-specific promoters. It has been proven that grafting can be an effective strategy for reducing heavy metal accumulation in the edible parts of plants while maintaining crop productivity [88]. This example supports our argument that both genetic and agronomic approaches can be leveraged to mitigate heavy metal stress in crop production systems. Additionally, overexpressing HIPPs in non-food plants may enable rhizofiltration, while integrating HIPP-mediated stress priming (or eustress/hormesis) into crop management could exploit controlled low-dose stress to pre-activate HIPP-dependent protective pathways [89]. Finally, the interplay between HIPP-mediated metal homeostasis and plant immunity should be dissected using dual-transcriptomic analyses performed during infection, in conjunction with metal-flux biosensors.

Figure 3: Schematic representation of the multifunctional roles of HIPP proteins in plants. The central structure shows the conserved N-terminal HMA domain (with the Cys-XX-Cys metal-binding motif) and the C-terminal CaaX isoprenylation motif, which anchors HIPPs to membranes. Four surrounding quadrants summarize the major biological functions: (1) heavy metal detoxification (e.g., Cd2+), leading to reduced metal accumulation and enhanced tolerance; (2) abiotic stress responses (drought, cold, salt, alkaline) through interactions with transcription factors and involvement in hormone signaling (e.g., ABA); (3) regulation of plant growth and development, including flowering, germination, spike development, and root elongation; and (4) plant–pathogen interactions, where HIPPs are targeted by pathogen effectors or act as resistance factors against bacterial pathogens. The schematic was constructed using BioGDP (www.BioGDP.com). Solid represents direct binding, and dotted represents hypothesized connections.