Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/ At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1independent mechanism to target peroxisomes for autophagic degradation.

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Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits. Key traits that distinguish these species are mediated by specialized cell types within organs. Here, we compare the transcriptomes of all cells within roots in three grasses—Zea mays (maize), Sorghum bicolor (sorghum), and outgroup Setaria viridis (Setaria). We first show that single-cell and single-nucleus RNA-seq provide complementary readouts of cell identity, warranting a combined analysis. Comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than others, in part by recruiting gene modules from other cell types. Furthermore, examining the whole genome duplication in maize, we detect extensive dosage compensation in surviving co-expressed homeologs, reinforcing genomic balance1. Homeolog pairs that underwent subfunctionalization2, partitioning their expression among cell types, represented a minor pattern but showed the highest rate of acquiring a novel (non-ancestral) domain. These results fit a conjecture in which mechanisms that maintain stoichiometric balance at the molecular level aid in homeolog retention for extended periods to allow new functions to arise. An unexpected synergy between spatial sub- and neo-functionalization then contributes to changes in transcriptional cell identity.

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