Rothstein, S. J. | bioRxiv Science

bioRxiv2026

Nucleoporin1 maintains male germ unit organization and transport in Arabidopsis pollen tubes, likely through shaping nuclear morphology

The male germ unit (MGU) in Arabidopsis pollen is comprised of one vegetative nucleus (VN) and two sperm nuclei (SN). It is evolutionarily specialized to deliver immotile sperm nuclei to an ovule for fertilization. Despite some progress in research on MGU, its organization and transport remain only partially understood. Here, we identified Nucleoporin1/136 as a new player in the structural organization and positioning of MGU in pollen tubes. We and others have previously reported the reduced fertility of nup1-1 plants; however, the mechanism remains unknown. In this work, we further examined the role of NUP1 in fertility using two mutant alleles, nup1-1 and nup1-2-/+. The reciprocal crosses between the nup1 mutants and the Col-0 wild type indicate that the nup1 mutant pollen is defective. To study the effect of a complete NUP1 knockout on pollen, we generated a transgenic line that produces pollen with and without NUP1 expression. This work led to the surprising discovery that the NUP1 protein is inherited from the pollen mother cell to the daughter cell during microgametophyte development. Subsequent in vitro experiments showed that NUP1 is required for pollen germination and pollen tube elongation. Further microscopic studies demonstrated that NUP1 is highly expressed in VN and essential for maintaining nuclear shape and size. We also demonstrated that NUP1 is required for proper MGU organization and transport, likely through maintaining VN morphology. Notably, our finding of nuclear morphology-mediated regulation of MGU may also explain the mechanistic details underlying the defective MGU movement in previously reported mutants such as kaku4, wit, and wip, which have abnormal nuclear morphology.

plant biology↗

bioRxiv2023

ENOD93 interacts with cytochrome c oxidase altering respiratory ATP production and root growth in plants

The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout of enod93 in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed in enod93. However, a progressive loss of ADP-dependent respiration rate was observed in enod93 mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of whole enod93 seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots of enod93 compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes. Highlight significanceWe identify the enigmatic early nodulin ENOD93 gene family as the plant homolog of the N-terminal regulatory domain of the yeast RESPIRATORY SUPERCOMPLEX 2 (RCF2) of the mitochondrial oxidative phosphorylation system and provide biochemical and physiological evidence of its role in plant ATP production, broadly explaining the role of ENOD93 in plants.

plant biology↗