Iron-dependent regulation of leaf senescence: a key role for the H2B histone variant HTB4

Iron is an essential micronutrient for plant growth and development, as well as for crop productivity and the quality of their derived products (Briat et al., 2015). This is because iron is a co-factor for several metalloproteins involved in essential physiological processes such as respiration in mitochondria or photosynthesis in chloroplasts. In most soils, iron is present in the form of insoluble oxides/hydroxides rendering it poorly available to plants. To cope with this poor bioavailability, plants have evolved sophisticated strategies to take up iron from soils (Berger et al., 2023; Li et al., 2023). Arabidopsis plants preferentially use the reduction-based strategy (the so-called Strategy I), as do most dicots and nongraminous monocots. This strategy relies on the secretion of protons into the rhizosphere by AHA2 (ARABIDOPSIS H⁺ ATPASE 2) to decrease the pH of the soil solution and solubilize oxidized iron (Fe³⁺), which is then reduced to Fe²⁺ by FRO2 (ferric reduction oxidases 2), and subsequently taken up into the root by IRT1 (iron-regulated transporter 1). This process is tightly regulated since, in excess, iron is also detrimental to the plant because of its capacity to generate reactive oxygen species (ROS) via the Fenton reaction.

The regulation of Iron homeostasis is well-conserved in plants, and is primarily controlled at the transcriptional level (Berger et al., 2023; Li et al., 2023). It relies on an intricate regulatory network involving several regulatory proteins, among which the bHLH (basic helix–loop–helix) transcription factors play a preponderant role (Fig. 1). For instance, Arabidopsis has 17 different bHLH proteins (from six bHLH clades) that regulate iron homeostasis. This regulatory network is composed of two modules. The first module relies on FIT/bHLH29 (FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR; clade IIIa). FIT is a direct positive regulator of FRO2 and IRT1 expression (Fig. 1). To achieve its function, FIT interacts with bHLH38, bHLH39, bHLH100, and bHLH101 (clade Ib), forming heterodimers with partially overlapping roles. In the second module, another set of bHLH transcription factors positively regulate the expression of FIT and clade Ib bHLHs (Fig. 1). It involves ILR3/bHLH105 (iaa-leucine resistant 3), IDT1/bHLH34 (iron deficiency tolerant 1), bHLH104, bHLH115 from clade IVc, and URI/bLHL121 (UPSTREAM REGULATOR OF IRT1) from clade IVb (Tissot et al., 2019; Gao et al., 2020). By contrast, PYE/bHLH47 (POPEYE; clade IVb) is a negative regulator of clade Ib bHLH expression (Pu & Liang, 2023).

In their study, Yang et al. demonstrated that the H2B histone variant HTB4 negatively regulates leaf senescence in an iron-dependent manner. For instance, HTB4 expression is induced under iron deficiency and is repressed under iron sufficiency. In addition, htb4 loss-of-function mutants display an early leaf senescence phenotype, reduced IRT1 and FRO2 expression, and decreased iron content. The early leaf senescence phenotype can be reverted by providing extra iron to the htb4 mutants, or by overexpressing IRT1. Further investigations provided the molecular mechanism by which HTB4 functions in regulating leaf senescence (Fig. 2). The authors showed that HTB4 promotes the expression of FIT and the four transcription factors of clade Ib bHLH. They further revealed that the binding of HTB4 to the promoter of clade Ib bHLHs correlates with an enrichment of the active mark H3K4me3 (histone H3 lysine 4 tri-methylation) in the vicinity of their transcriptional start sites. As a result, the expression of clade Ib bHLHs is promoted, and in turn, the expression of IRT1 and FRO2, fostering root iron uptake and delaying leaf senescence.

Among the genes whose expression was decreased in the htb4 mutant when compared to wild-type plants (transcriptome data analysis), some have been described as playing a key role in regulating iron homeostasis. This is the case for the above-mentioned IDT1 bHLH transcription factor, which is a direct positive regulator of clade Ib bHLHs expression. This is also the case for IMA2/FEP2 (IRONMAN 2/FE-UPTAKE-INDUCING PEPTIDE 2) and IMA3/FEP1, which encode peptides that are positive regulators of the iron deficiency response. These peptides inhibit the activity of hemerythrin E3 ubiquitin ligases (i.e. BTS, BRUTUS; BTSL1 and 2, BRUTUS LIKE 1 and 2) that negatively regulate iron homeostasis by promoting the ubiquitin-mediated degradation via the 26S proteasome, of ILR3 and bHLH115 (i.e. BTS), and FIT (i.e. BTSL1 and BTSL2; Selote et al., 2015; Rodríguez-Celma et al., 2019; Li et al., 2021). These observations suggest that HTB4 might also directly regulate the expression of additional genes involved in the transcriptional regulation of iron homeostasis, notably those upstream from FIT and clade Ib bHLHs (Fig. 1). Conversely, it would also be interesting to investigate further if a negative correlation exists between HTB4 and genes that encode proteins that negatively regulate clade Ib expression either directly, such as PYE, or indirectly such as BTS, BTSL1, and BTSL2 (Rodríguez-Celma et al., 2019; Pu & Liang, 2023).

Yang et al. showed that root iron uptake is impaired in htb4 because the expression of IRT1 and FRO2 was decreased. Whether other mechanisms related to the maintenance of iron homeostasis (e.g. iron translocation, storage, or assimilation) are regulated in an HTB4-dependent manner is also an open question. For instance, the expression of NAS1 (nicotianamine synthase 1), NAS4 (nicotianamine synthase 4), and OPT3 (oligo peptide transporter 3) is reduced in htb4 (transcriptome analysis). These three genes participate in the translocation and partitioning of iron between the plant tissues via the phloem, which suggests that these functions are also regulated by HTB4. Moreover, OPT3 is also involved in communicating the shoot iron status to the root to balance the uptake of iron with the plant needs (Mendoza-Cózatl et al., 2014).

At neutral to alkaline pH, iron solubility in soils is low and the activity of ferric reductases, such as FRO2, is strongly impaired (Susin et al., 1996). To adapt to such soils, Arabidopsis plants secrete into the rhizosphere iron mobilizing coumarins (i.e. fraxetin; Robe et al., 2021a,b). It was recently proposed that once secreted, fraxetin would mostly form Fe³⁺-fraxetin complexes rather than reducing Fe³⁺ into Fe²⁺, indicating that the IRT1/FRO2 system does not play a major role in the uptake of iron at alkaline pH. Instead, it is suggested that Fe³⁺-fraxetin complexes are directly taken up into the plant root via a yet unknown mechanism (Robe et al., 2021c). Whether or not in such soil environments HTB4 plays a role in modulating iron uptake/homeostasis, and thus leaf senescence, deserves to be investigated.

Last but not least, it would be of interest to determine if the role of HTB4 in delaying leaf senescence is conserved in grass species such as rice (Oriza sativa) since the uptake of iron mostly relies on an IRT1/FRO2-independent mechanism (Li et al., 2021).

In conclusion, the study of Yang et al. provides a molecular framework by which mineral nutrients such as iron can interfere with leaf senescence. It also opens the road for new investigations to determine the extent of this process either with iron or with other mineral nutrients.