Cytokinins are hormones that play an essential role in plant growth and development. The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels. Cytokinin oxidase has been well characterized biochemically, but its regulation at the molecular level is not well understood. We isolated a cytokinin oxidase open reading frame from maize (Zea mays), called Ckx1, and we used it as a probe in northern and in situ hybridization experiments. We found that the gene is expressed in a developmental manner in the kernel, which correlates with cytokinin levels and cytokinin oxidase activity. In situ hybridization with Ckx1 and transgenic expression of a transcriptional fusion of the Ckx1 promoter to the Escherichia coli beta-glucuronidase reporter gene revealed that the gene is expressed in the vascular bundles of kernels, seedling roots, and coleoptiles. We show that Ckx1 gene expression is inducible in various organs by synthetic and natural cytokinins. Ckx1 is also induced by abscisic acid, which may control cytokinin oxidase expression in the kernel under abiotic stress. We hypothesize that under non-stress conditions, cytokinin oxidase in maize plays a role in controlling growth and development via regulation of cytokinin levels transiting in the xylem. In addition, we suggest that under environmental stress conditions, cytokinin oxidase gene induction by abscisic acid results in aberrant degradation of cytokinins therefore impairing normal development.

Two maize genes with predicted translational similarity to the Arabidopsis FIE (Fertilization-Independent Endosperm) protein, a repressor of endosperm development in the absence of fertilization, were cloned and analyzed. Genomic sequences of fie1 and fie2 show significant homology within coding regions but none within introns or 5 upstream. The fie1 gene is expressed exclusively in the endosperm of developing kernels starting at ‫ف‬ 6 days after pollination. fie1 is an imprinted gene showing no detectable expression of the paternally derived fie1 allele during kernel development. Conversely, fie2 is expressed in the embryo sac before pollination. After pollination, its expression persists, predominantly in the embryo and at lower levels in the endosperm. The paternal fie2 allele is not expressed early in kernel development, but its transcription is activated at 5 days after pollination. fie2 is likely to be a functional ortholog of the Arabidopsis FIE gene, whereas fie1 has evolved a distinct function. The maize FIE2 and sorghum FIE proteins form a monophyletic group, sharing a closer relationship to each other than to the FIE1 protein, suggesting that maize fie genes originated from two different ancestral genomes.

The maize lethal leaf spot 1 (lls1) mutant exhibits enhanced resistance to fungal pathogens. The lls1 resistance to Cochliobolus heterostrophus has two components: (i) lesion number is reduced 40% relative to wild type; and (ii) the lesions that do form often do not contain viable fungus. This lesion sterility is dependent upon leaf maturity and light, whereas reduced lesion number is not. The lls1 lesions express pathogenesis-related proteins at high levels, so lesion sterility likely results from activation of defense systems and necrosis. Reduced lesion number is correlated with a reduction of C. heterostrophus spore germination, hyphal growth, and haustoria formation on the leaf epidermis. The rust pathogen Puccinia sorghi has reduced pustule formation on lls1, and its germination and growth are also slowed on the epidermis. However, after entering the mesophyll through stomata, P. sorghi can form pustules on lls1, and even green islands within necrotic lls1 lesions. In situ mRNA hybridization shows that Lls1 is predominantly expressed in the leaf epidermis, coincident with the site of resistance in the mutant.

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