Zhubing Hu

Phosphate (Pi) deficiency promotes premature leaf senescence in plants through complex molecular networks; however, the underlying mechanisms remain poorly understood. Inositol polyphosphate 5‐phosphatases (5PTases) are key regulators of inositol phosphate metabolism, but the maize ( Zea mays L.) 5PTase gene family and its roles in low Pi (LP)‐induced leaf senescence have not been systematically investigated. Here, we identified 31 members of the Zm5PTase family in the maize genome. Phylogenetic and structural analyses revealed marked divergence among clades. Among these genes, Zm5PTase25 was preferentially expressed in senescing leaves and significantly downregulated under LP stress. Functional analyses indicated that Zm5PTase25 negatively regulates LP‐induced leaf senescence. Silencing Zm5PTase25 accelerated leaf senescence, promoted anthocyanin accumulation, altered Pi distribution between old and young leaves in a manner consistent with enhanced Pi remobilisation, and upregulated senescence‐ and ribonuclease‐associated genes and repressed photosynthesis‐related genes. Conversely, overexpression of Zm5PTase25 delayed leaf senescence. In vitro assays showed that Zm5PTase25 bound and hydrolysed soluble InsP ₃ and InsP ₄ . The PHR1‐like transcription factor ZmPHL6 was identified as a potential upstream transcriptional repressor of Zm5PTase25 . Moreover, silencing ZmPHL6 produced phenotypes opposite to those of Zm5PTase25 ‐silenced plants. Collectively, our findings suggest that the ZmPHL6 ‐ Zm5PTase25 regulatory module may modulate LP‐induced leaf senescence, potentially by influencing source to sink Pi remobilisation, providing insights into molecular mechanisms coordinating leaf senescence with maize adaptation to Pi starvation.

SUMMARY: Pumilio (PUM) proteins comprise an evolutionarily conserved family of sequence‐specific RNA‐binding proteins that act as key post‐transcriptional regulators in eukaryotes. In this study, we identified a maize PUM protein, named ZmDLR9 (Defective in Lateral Root 9), as a positive factor involved in lateral root development. Loss‐of‐function mutation in the ZmDLR9 ( dlr9‐1 ) severely impaired lateral root formation. Functional analysis revealed that ZmDLR9 localizes in the nucleolus and interacts with pre‐rRNA processing‐associated proteins, including BRX1‐1 (BIOGENESIS OF RIBOSOMES IN XENOPUS 1–1), BRX1‐2, and GRC3 (a subunit of the ITS2 processing complex). The dlr9‐1 mutation resulted in aberrant accumulation of pre‐rRNA processing intermediates, as well as defects in ribosome assembly and translation. Heterologous expression of ZmDLR9 partially rescued the growth defects of an Arabidopsis PUM loss‐of‐function mutant ( apum24‐2 ), demonstrating functional conservation of the PUM protein family across the plant kingdom. Furthermore, the dlr9‐1 mutation disrupted nucleolar morphology, thereby inhibiting cell proliferation and delaying lateral root primordia emergence. Together, our results demonstrate that ZmDLR9, a Pumilio protein, is required for lateral root development by mediating pre‐rRNA processing‐dependent cell proliferation.

During development, cell fate determination hinges on the dynamic activities of multiple transcription factors (TFs), but how the activities of individual TFs contribute to developmental diversity remains incompletely understood. Here, we report that BZU2/ZmMUTE is responsible for the functional and spatial heterogeneity of the four-celled stomatal complexes in maize (Zea mays), in part through its liquid–liquid phase separation (LLPS) property. Genotypic and single-cell RNA-seq analyses confirmed previous findings that ZmMUTE is associated with lineage-specific gene expression during stomatal development, while providing additional resolution of cell-type-specific transcriptional programs. The intrinsically disordered region 4 (IDR4) promotes the formation of ZmMUTE condensates, which can recruit the ZmMUTE partner ZmSCRM and associate with transcriptionally active sites. These condensates are associated with enhanced DNA binding and target gene activation, potentially influencing the development of guard cells and subsidiary cells. IDR4 is also sufficient for conferring cell-to-cell mobility to AtMUTE, highlighting its conserved role in facilitating intercellular movement. Taken together, the dynamic behavior of ZmMUTE, owing to its condensational properties, appears to orchestrate cell-type-specific regulation, thereby enabling the emergence of the unique four-celled stomatal morphology.

Lateral roots (LRs), the primary component of the maize root system, are crucial for water and nutrient acquisition. Deciphering the molecular mechanisms underlying LR formation and development is therefore essential for improving maize yield and stress resilience. In this study, we characterised a mutant, defective in lateral root 2‐1 (dlr2‐1), which displays impaired LR development and compromised drought resistance. Phenotypic analysis and RNA‐seq revealed defective cell proliferation in lateral root primordia (LRP) of dlr2‐1, likely attributable to DNA damage. Accordingly, the dlr2‐1 mutant exhibited an activated DNA damage response. Exogenous treatment of wild‐type B73 plants with genotoxic agents recapitulated the dlr2‐1 phenotype, suppressing LRP emergence and reducing mature LR numbers. Positional cloning and allelic analysis pinpointed a missense mutation in DLR2, causing a leucine‐to‐glutamine substitution at residue 1035 (ZmDLR2^L1035Q) and accounting for the LR defects in dlr2‐1. ZmDLR2 encodes a maize orthologue of Arabidopsis BRUSHY1 (BRU1), a key regulator of DNA damage repair during DNA replication. Comet assays demonstrated that dlr2‐1 accumulates more severe DNA fragmentation than B73, as evidenced by an elevated tail moment, which was further aggravated under genotoxic stress. Moreover, ZmDLR2 mutation adversely affected plant height and kernel size in dlr2 mutants, leading to significant yield reduction. Collectively, our results establish that ZmDLR2 is indispensable for DNA repair and that its dysfunction activates the DNA damage response, ultimately inhibiting cell proliferation, disrupting LRP initiation and LR formation and compromising maize productivity.

Chlorophyll biosynthesis must be tightly coupled to light-harvesting chlorophyll a/b-binding protein (LHCP) biogenesis, as free chlorophyll and its precursors are phototoxic. However, precisely how these 2 processes are coordinated in Arabidopsis (Arabidopsis thaliana) remains elusive. Our previous studies demonstrated the role of LHCP TRANSLOCATION DEFECT (LTD) in delivering LHCPs to the chloroplast via the signal recognition particle-dependent pathway. Here, we show that LTD interacts with and stabilizes the chlorophyll biosynthesis enzymes Mg-protoporphyrin methyltransferase and Mg-protoporphyrin monomethylester (MgPME) cyclase, maintaining their activity. We also demonstrate the direct binding of LTD to MgPME, and through crystal structure analysis, we show that the groove of the LTD dimer is critical for MgPME binding. Thus, we propose that LTD transfers MgPME from Mg-protoporphyrin methyltransferase to the MgPME cyclase. These results elucidate a role for LTD in synchronizing chlorophyll biosynthesis with LHCP transport to ensure the correct insertion of chlorophylls into LHCPs.

Root System Architecture (RSA) is a crucial plant trait that governs a plant's ability to absorb water and nutrients. In this study, we describe a mutant with nutrient‐dependent defects in root development, affecting both the primary root and lateral roots (LRs). This mutant, identified through a screen for defects in LR development, has been designated dlr1‐1. The dlr1‐1 mutant exhibits impaired LR emergence rather than defects in the LR primordium (LRP) formation, particularly under potassium (K⁺)‐deprivation conditions. This impairment likely stems from inhibited cell proliferation caused by the dlr1‐1 mutation. K⁺ deprivation specifically leads to the accumulation of salicylic acid (SA) in the dlr1‐1 mutant, consistent with the upregulation of SA biosynthesis genes. Moreover, exogenous application of SA to wild‐type plants (B73) mimics the dlr1‐1 phenotype. Conversely, treatment of the dlr1‐1 mutant with 2‐aminoindane‐2‐phosphonic acid, an SA biosynthesis inhibitor, partially restores LR emergence, indicating that elevated SA levels may be responsible for the mutant's developmental defects. MutMap analysis and allelism tests confirmed that the phenotypes of the dlr1‐1 mutant results from the loss of the Na⁺/H⁺ antiporter, ZmNHX7. Additionally, the application of NaCl exacerbates the dlr1‐1 mutant phenotype, suggesting that the root defects in dlr1‐1 mutant depend on ion homoeostasis. In conclusion, our findings demonstrate that maize DLR1/NHX7 is essential for root development under potassium deprivation.

In Arabidopsis, RNA editing alters more than 500 cytidines (C) to uridines (U) in mitochondrial transcripts, a process involving the family of pentatricopeptide repeat (PPR) proteins. Here, we report a previously uncharacterized mitochondrial PLS-type PPR protein, GEND2, which functions in the mitochondrial RNA editing. The T-DNA insertion in the 5′-untranslated region of GEND2, referred to as gend2-1, results in defective root development compared to wild-type (WT) plants. A comprehensive examination of mitochondrial RNA editing sites revealed a significant reduction in the gend2-1 mutant compared to WT plants, affecting six specific mitochondrial RNA editing sites, notably within the mitochondrial genes CcmFn-1, RPSL2 and ORFX. These genes encode critical components of cytochrome protein maturation pathway, mitochondrial ribosomal subunit, and twin arginine translocation subunits, respectively. Further analysis of the transcriptional profile of the gend2-1 mutant and wild type revealed a striking induction of expression in a cluster of genes associated with mitochondrial dysfunction and regulated by ANAC017, a key regulator coordinating organelle functions and stress responses. Intriguingly, the gend2-1 mutation activated an ANAC017-dependent signaling aimed at countering cell wall damage induced by cellulose synthase inhibitors, as well as an ANAC017-independent pathway that retarded root growth under normal condition. Collectively, our findings identify a novel mitochondrial PLS-type PPR protein GEND2, which participates in the editing of six specific mitochondrial RNA editing sites. Furthermore, the gend2-1 mutation triggers two distinct pathways in plants: an ANAC017-dependent pathway and ANAC017-independent pathway.