Cytosolic malate dehydrogenase (MDH) is a key enzyme that regulates the interconversion between malate and oxaloacetate (OAA). However, its role in modulating storage compound accumulation in maize endosperm is largely unknown. Here, we characterized a novel naturally occurring maize mdh4-1 mutant, which produces small, opaque kernels and exhibits reduced starch but enhanced lysine content. Map-based cloning, functional complementation and allelism analyses identified ZmMdh4 as the causal gene. Enzymatic assays demonstrated that ZmMDH4 predominantly catalyses the conversion from OAA to malate. In comparison, the activity of the mutant enzyme, which lacks one glutamic acid (Glu), was completed abolished, demonstrating that the Glu residue was essential for ZmMDH4 function. Knocking down ZmMdh4 in vivo led to a substantial metabolic shift towards glycolysis and a dramatic disruption in the activity of the mitochondrial complex I, which was correlated with transcriptomic alterations. Taken together, these results demonstrate that ZmMdh4 regulates the balance between mitochondrial respiration and glycolysis, ATP production and endosperm development, through a yet unknown feedback regulatory mechanism in mitochondria.

Heterosis has been widely used to increase grain quality and yield, but its genetic mechanism remains unclear. In this study, the genetic basis of heterosis for four maize kernel traits was examined in two test populations constructed using a set of 184 chromosome segment substitution lines (CSSLs) and two inbred lines (Zheng58 and Xun9058) in two environments. 63 and 57 different heterotic loci (HL) were identified for four kernel traits in the CSSLs × Zheng58 and CSSLs × Xun9058 populations, respectively. Of these, nine HL and six HL were identified for four kernel traits in the CSSLs × Zheng58 and CSSLs × Xun9058 populations, at the two locations simultaneously. Comparative analysis of the HL for the four kernel traits identified only 21 HL in the two test populations simultaneously. These results showed that most HL for the four kernel traits differed between the two test populations. The common HL were important loci from the Reid × Tangsipingtou heterotic model, and could be used to predict hybrid performance in maize breeding.

Summary Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map‐based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α‐tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin‐containing TCP‐1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus‐end binding protein END‐BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species‐specific divergence.

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