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 β-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.

Drought stress is one of the main environmental problems encountered by crop growers. Reduction in arable land area and reduced water availability make it paramount to identify and develop strategies to allow crops to be more resilient in water-limiting environments. The plant hormone abscisic acid (ABA) plays an important role in the plants’ response to drought stress through its control of stomatal aperture and water transpiration, and transgenic modulation of ABA levels therefore represents an attractive avenue to improve the drought tolerance of crops. Several steps in the ABA-signaling pathway are controlled by ubiquitination involving really interesting new genes (RING) domain-containing proteins. We characterized the maize (Zea mays) RING protein family and identified two novel RING-H2 genes called ZmXerico1 and ZmXerico2. Expression of ZmXerico genes is induced by drought stress, and we show that overexpression of ZmXerico1 and ZmXerico2 in Arabidopsis and maize confers ABA hypersensitivity and improved water use efficiency, which can lead to enhanced maize yield performance in a controlled drought-stress environment. Overexpression of ZmXerico1 and ZmXerico2 in maize results in increased ABA levels and decreased levels of ABA degradation products diphaseic acid and phaseic acid. We show that ZmXerico1 is localized in the endoplasmic reticulum, where ABA 8′-hydroxylases have been shown to be localized, and that it functions as an E3 ubiquitin ligase. We demonstrate that ZmXerico1 plays a role in the control of ABA homeostasis through regulation of ABA 8′-hydroxylase protein stability, representing a novel control point in the regulation of the ABA pathway.

Leaf morphogenesis requires growth polarized along three axes—proximal-distal (P-D) axis, medial-lateral axis, and abaxial-adaxial axis. Grass leaves display a prominent P-D polarity consisting of a proximal sheath separated from the distal blade by the auricle and ligule. Although proper specification of the four segments is essential for normal morphology, our knowledge is incomplete regarding the mechanisms that influence P-D specification in monocots such as maize (Zea mays). Here, we report the identification of the gene underlying the semidominant, leaf patterning maize mutant Hairy Sheath Frayed1 (Hsf1). Hsf1 plants produce leaves with outgrowths consisting of proximal segments—sheath, auricle, and ligule—emanating from the distal blade margin. Analysis of three independent Hsf1 alleles revealed gain-of-function missense mutations in the ligand binding domain of the maize cytokinin (CK) receptor Z. mays Histidine Kinase1 (ZmHK1) gene. Biochemical analysis and structural modeling suggest the mutated residues near the CK binding pocket affect CK binding affinity. Treatment of the wild-type seedlings with exogenous CK phenocopied the Hsf1 leaf phenotypes. Results from expression and epistatic analyses indicated the Hsf1 mutant receptor appears to be hypersignaling. Our results demonstrate that hypersignaling of CK in incipient leaf primordia can reprogram developmental patterns in maize.

Leaf morphogenesis requires growth polarized along three axes - proximal-distal, medial-lateral and abaxial-adaxial. Grass leaves display a prominent proximal-distal (P-D) polarity consisting of a proximal sheath separated from the distal blade by the auricle and ligule. Although proper specification of the four segments is essential for normal morphology, our knowledge is incomplete regarding the mechanisms which influence P-D specification in monocots like maize ( Zea mays ). Here we report the identification of the gene underlying the semi-dominant, leaf patterning, maize mutant Hairy Sheath Frayed1 ( Hsf1 ). Hsf1 plants produce leaves with outgrowths consisting of proximal segments – sheath, auricle and ligule – emanating from the distal blade margin. Analysis of three independent Hsf1 alleles revealed gain-of-function missense mutations in the ligand binding domain of the maize cytokinin (CK) receptor Zea mays Histidine Kinase1 ( ZmHK1) gene. Biochemical analysis and structural modeling suggest the mutated residues near the CK binding pocket affect CK binding affinity. Treatment of wild type seedlings with exogenous CK phenocopied the Hsf1 leaf phenotypes. Results from expression and epistatic analyses indicated the Hsf1 mutant receptor appears to be hypersignaling. Our results demonstrate that hypersignaling of CK in incipient leaf primordia can reprogram developmental patterns in maize. Summary: Increased cytokinin signaling in the maize Hairy Sheath Frayed1 mutant modifies leaf development leading to changes in pattering, growth and cell identity.

Key message : CRISPR-Cas multiplex editing generated a broad range of early-flowering soybean lines, advancing flowering by up to four weeks under controlled long-day conditions and by approximately one to two weeks in a field trial. Soybean is a promising crop for European agriculture, but its cultivation area remains smaller than its potential. A major limitation to broader adoption in Europe is the restricted number of elite lines adapted to local environmental conditions, particularly the photoperiod encountered at higher latitude. At these latitudes, soybean must flower early to complete its growth cycle before the onset of harsh autumn conditions. In this project, we employed a CRISPR-Cas multiplex knockout strategy, to generate allelic diversity in the gene network controlling flowering time, with the aim of developing earlier flowering soybeans lines better suited to European high latitudes. This approach successfully produced earlier flowering varieties, with flowering advances of up to four weeks compared to the controls. Starting from maturity group (MG) I and MG III lines, we developed, within two generations, gene-edited early flowering soybean lines with estimated maturity ranges between MG 000 and MG 0. Greenhouse observations were supported by a field trial, in an MG II environment, where edited lines flowered approximately one to two weeks earlier than their respective parental controls. These results demonstrate that multiplex editing of flowering-time genes can rapidly generate targeted allelic diversity and a broad spectrum of flowering phenotypes within elite soybean backgrounds. These results provide valuable breeding material and a genotype/phenotype resource for subsequent evaluation of flowering-time adaptation across environments.