zmm28 transgenic maize increases both N uptake- and N utilization-efficiencies.

Biotechnology has emerged as a valuable tool in the development of maize (Zea mays L.) hybrids with enhanced nitrogen (N) use efficiency. Recent work has described the positive effects of an increased and extended expression of the zmm28 transcription factor (Event DP202216) on maize yield productivity. In this study, we expand on the previous findings studying maize N uptake and utilization in DP202216 transgenic hybrids compared to wild-type (WT) controls. Isotope 15N labeling demonstrates that DP202216 hybrids have an improved N uptake during late-vegetative stages (inducing N storage in lower leaves of the canopy) and, thus, N uptake efficiency (N uptake to applied N ratio) relative to WT. Through both greater N harvest index and reproductive N remobilization, transgenic plants were able to achieve better N utilization efficiency (yield to N uptake ratio). Our findings suggest the DP202216 trait could open new avenues for improving N uptake and utilization efficiencies in maize.

KIL1 terminates fertility in maize by controlling silk senescence.

Plant flowers have a functional life span during which pollination and fertilization occur to ensure seed and fruit development. Once flower senescence is initiated, the potential to set seed or fruit is irrevocably lost. In maize, silk strands are the elongated floral stigmas that emerge from the husk-enveloped inflorescence to intercept airborne pollen. Here we show that KIRA1-LIKE1 (KIL1), an ortholog of the Arabidopsis NAC (NAM (NO APICAL MERISTEM), ATAF1/2 (Arabidopsis thaliana Activation Factor1 and 2) and CUC (CUP-SHAPED COTYLEDON 2)) transcription factor KIRA1, promotes senescence and programmed cell death (PCD) in the silk strand base, ending the window of accessibility for fertilization of the ovary. Loss of KIL1 function extends silk receptivity and thus strongly increases kernel yield following late pollination. This phenotype offers new opportunities for possibly improving yield stability in cereal crops. Moreover, despite diverging flower morphologies and the substantial evolutionary distance between Arabidopsis and maize, our data indicate remarkably similar principles in terminating floral receptivity by PCD, whose modulation offers the potential to be widely used in agriculture.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize.

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

Successes and insights of an industry biotech program to enhance maize agronomic traits.

Corteva Agriscience™ ran a discovery research program to identify biotech leads for improving maize Agronomic Traits such as yield, drought tolerance, and nitrogen use efficiency. Arising from many discovery sources involving thousands of genes, this program generated over 3331 DNA cassette constructs involving a diverse set of circa 1671 genes, whose transformed maize events were field tested from 2000 to 2018 under managed environments designed to evaluate their potential for commercialization. We demonstrate that a subgroup of these transgenic events improved yield in field-grown elite maize breeding germplasm. A set of at least 22 validated gene leads are identified and described which represent diverse molecular and physiological functions. These leads illuminate sectors of biology that could guide crop improvement in maize and perhaps other crops. In this review and interpretation, we share some of our approaches and results, and key lessons learned in discovering and developing these maize Agronomic Traits leads.

Knockouts of a late flowering gene via CRISPR-Cas9 confer early maturity in rice at multiple field locations.

KEY MESSAGE: OsGhd7 gene was discovered by screening our rice activation tagging population. CRISPR-Cas9 created knockouts of OsGhd7 conferred early flowering and early maturity in rice varieties across multiple geographical locations in China. Our research shows that OsGhd7 is a good target for breeding early maturity rice varieties, and an excellent example of the advantages of applying the CRISPR-Cas9 technology for trait improvement. Flowering time (heading date) is an important trait for crop cultivation and yield. In this study, we discovered a late flowering gene OsGhd7 by screening our rice activation tagging population, and demonstrated that overexpression of OsGhd7 delayed flowering time in rice, and the delay in flowering time depended on the transgene expression level. OsGhd7 is a functional allele of the Ghd7 gene family; knockouts of OsGhd7 generated by CRISPR-Cas9 significantly accelerated flowering time and the earliness of the flowering time depended on field location. The homozygous OsGhd7 knockout lines showed approximately 8, 10, and 20 days earlier flowering than controls at three different locations in China (Changsha City, Sanya City, and Beijing City, respectively) that varied from 18.25° N to 39.90° N. Furthermore, knockouts of OsGhd7 also showed an early flowering phenotype in different rice varieties, indicating OsGhd7 can be used as a common target gene for using the CRISPR technology to modulate rice flowering time. The importance of OsGhd7 and CRISPR technology for breeding early maturity rice varieties are discussed.

Maize BIG GRAIN1 homolog overexpression increases maize grain yield.

The Zea Mays BIG GRAIN 1 HOMOLOG 1 (ZM-BG1H1) was ectopically expressed in maize. Elite commercial hybrid germplasm was yield tested in diverse field environment locations representing commercial models. Yield was measured in 101 tests across all 4 events, 26 locations over 2 years, for an average yield gain of 355 kg/ha (5.65 bu/ac) above control, with 83% tests broadly showing yield gains (range +2272 kg/ha to -1240 kg/ha), with seven tests gaining more than one metric ton per hectare. Plant and ear height were slightly elevated, and ear and tassel flowering time were delayed one day, but ASI was unchanged, and these traits did not correlate to yield gain. ZM-BG1H1 overexpression is associated with increased ear kernel row number and total ear kernel number and mass, but individual kernels trended slightly smaller and less dense. The ZM-BG1H1 protein is detected in the plasma membrane like rice OS-BG1. Five predominant native ZM-BG1H1 alleles exhibit little structural and expression variation compared to the large increased expression conferred by these ectopic alleles.

A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice.

KEY MESSAGE: CRISPR-edited variants at the 3'-end of OsLOGL5's coding sequence (CDS), significantly increased rice grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Cytokinins impact numerous aspects of plant growth and development. This study reports that constitutive ectopic overexpression of a rice cytokinin-activation enzyme-like gene, OsLOGL5, significantly reduced primary root growth, tiller number, and yield. Conversely, mutations at the 3'-end of OsLOGL5 CDS resulted in normal rice plant morphology but with increased grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Six gene edited variants (Edit A to F) were created and tested in the field. Edit-B and Edit-F plants increased, but Edit-D and Edit-E plants decreased, the panicle number per plant. All OsLOGL5-edited plants significantly increased seed setting rate, total grain numbers, full-filled grain numbers per panicle, and thousand seed weight under drought conditions, suggesting that OsLOGL5 is likely involved in the regulation of both seed development and grain filling processes. Our results indicate that the C-terminal end of OsLOGL5 protein plays an important role in regulating rice yield improvement under different abiotic stress conditions, and OsLOGL5 is important for rice yield enhancement and stability.

Overexpression of zmm28 increases maize grain yield in the field.

Increasing maize grain yield has been a major focus of both plant breeding and genetic engineering to meet the global demand for food, feed, and industrial uses. We report that increasing and extending expression of a maize MADS-box transcription factor gene, zmm28, under the control of a moderate-constitutive maize promoter, results in maize plants with increased plant growth, photosynthesis capacity, and nitrogen utilization. Molecular and biochemical characterization of zmm28 transgenic plants demonstrated that their enhanced agronomic traits are associated with elevated plant carbon assimilation, nitrogen utilization, and plant growth. Overall, these positive attributes are associated with a significant increase in grain yield relative to wild-type controls that is consistent across years, environments, and elite germplasm backgrounds.

Over-expression of the photoperiod response regulator ZmCCT10 modifies plant architecture, flowering time and inflorescence morphology in maize.

Maize originated as a tropical plant that required short days to transition from vegetative to reproductive development. ZmCCT10 [CO, CONSTANS, CO-LIKE and TIMING OF CAB1 (CCT) transcription factor family] is a regulator of photoperiod response and was identified as a major QTL controlling photoperiod sensitivity in maize. We modulated expression of ZmCCT10 in transgenic maize using two constitutive promoters with different expression levels. Transgenic plants over expressing ZmCCT10 with either promoter were delayed in their transition from vegetative to reproductive development but were not affected in their switch from juvenile-to-adult vegetative growth. Strikingly, transgenic plants containing the stronger expressing construct had a prolonged period of vegetative growth accompanied with dramatic modifications to plant architecture that impacted both vegetative and reproductive traits. These plants did not produce ears, but tassels were heavily branched. In more than half of the transgenic plants, tassels were converted into a branched leafy structure resembling phyllody, often composed of vegetative plantlets. Analysis of expression modules controlling the floral transition and meristem identity linked these networks to photoperiod dependent regulation, whereas phase change modules appeared to be photoperiod independent. Results from this study clarified the influence of the photoperiod pathway on vegetative and reproductive development and allowed for the fine-tuning of the maize flowering time model.

Ectopic expression of ARGOS8 reveals a role for ethylene in root-lodging resistance in maize.

Ethylene plays a critical role in many diverse processes in plant development. Recent studies have demonstrated that overexpression of the maize ARGOS8 gene reduces the plant's response to ethylene by decreasing ethylene signaling and enhances grain yield in transgenic maize plants. The objective of this study was to determine the effects of ethylene on the development of nodal roots, which are primarily responsible for root-lodging resistance in maize. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was found to promote the emergence of nodal roots. Transcriptome analysis of nodal tissues revealed that the expression of genes involved in metabolic processes and cell wall biogenesis was upregulated in response to ACC treatment, supporting the notion that ethylene is a positive regulator for the outgrowth of young root primordia. In BSV::ARGOS8 transgenic plants with reduced ethylene sensitivity due to constitutive overexpression of ARGOS8, nodal root emergence was delayed and the promotional effect of ACC on nodal root emergence decreased. Field tests showed that the BSV::ARGOS8 plants had higher root lodging relative to non-transgenic controls. When ARGOS8 expression was controlled by the developmentally regulated promoter FTM1, which conferred ARGOS8 overexpression in adult plants but not in the nodal roots and nodes in juvenile plants, the FTM1::ARGOS8 plants had no significant difference in root lodging compared with the wild type but produced a higher grain yield. These results suggest that ethylene has a role in promoting nodal root emergence and that a delay in nodal root development has a negative effect on root-lodging resistance in maize.

Overexpression of RING Domain E3 Ligase ZmXerico1 Confers Drought Tolerance through Regulation of ABA Homeostasis.

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.

ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions.

Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas-enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought-tolerant crops.

Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction.

The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize.

Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize.

Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions.

Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought-stress conditions.

A transgenic gene-silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down-regulated ACC synthases (ACSs), enzymes that catalyse the rate-limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought-stress and rain-fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype × transgene) × environment interaction existed among the events, highlighting the need to better understand the context in which the down-regulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis-silking interval and a concomitant increase in kernel number/ear in transgene-positive events versus nulls. Selected events were also field tested under a low-nitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/acre) yield increase. This set of extensive field evaluations demonstrated that down-regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.

Maize global transcriptomics reveals pervasive leaf diurnal rhythms but rhythms in developing ears are largely limited to the core oscillator.

BACKGROUND: Plant diurnal rhythms are vital environmental adaptations to coordinate internal physiological responses to alternating day-night cycles. A comprehensive view of diurnal biology has been lacking for maize (Zea mays), a major world crop. METHODOLOGY: A photosynthetic tissue, the leaf, and a non-photosynthetic tissue, the developing ear, were sampled under natural field conditions. Genome-wide transcript profiling was conducted on a high-density 105 K Agilent microarray to investigate diurnal rhythms. CONCLUSIONS: In both leaves and ears, the core oscillators were intact and diurnally cycling. Maize core oscillator genes are found to be largely conserved with their Arabidopsis counterparts. Diurnal gene regulation occurs in leaves, with some 23% of expressed transcripts exhibiting a diurnal cycling pattern. These transcripts can be assigned to over 1700 gene ontology functional terms, underscoring the pervasive impact of diurnal rhythms on plant biology. Considering the peak expression time for each diurnally regulated gene, and its corresponding functional assignment, most gene functions display temporal enrichment in the day, often with distinct patterns, such as dawn or midday preferred, indicating that there is a staged procession of biological events undulating with the diurnal cycle. Notably, many gene functions display a bimodal enrichment flanking the midday photosynthetic maximum, with an initial peak in mid-morning followed by another peak during the afternoon/evening. In contrast to leaves, in developing ears as few as 47 gene transcripts are diurnally regulated, and this set of transcripts includes primarily the core oscillators. In developing ears, which are largely shielded from light, the core oscillator therefore is intact with little outward effect on transcription.

Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation.

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.

A member of the maize isopentenyl transferase gene family, Zea mays isopentenyl transferase 2 (ZmIPT2), encodes a cytokinin biosynthetic enzyme expressed during kernel development. Cytokinin biosynthesis in maize.

Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8-10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants.

Maize ribosome-inactivating proteins (RIPs) with distinct expression patterns have similar requirements for proenzyme activation.

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are potent naturally occurring toxins found in numerous and diverse plant species. The maize RIP is unusual among the plant RIPs because it is synthesized as an inactive precursor (also known as maize proRIP1 or b-32). The proenzyme undergoes proteolytic activation that results in the removal of the NH(2)-terminal, the COOH-terminal, and internal sequences to form a two-chain holoenzyme capable of irreversibly modifying the large rRNA. The characterization of a second maize RIP (RIP2), encoded by the gene designated Rip3:2 is described here. Low levels of Rip3:2 RNA were detected in roots, shoots, tassels, silks, and leaves, but the Rip3:2 gene, unlike the Rip3:1 gene, is not under the control of the transcriptional activator Opaque-2. Instead, its expression was up-regulated by drought. Rip3:2 encodes a 31.1 kDa polypeptide that is very similar to proRIP1 in regions corresponding to those found in the active protein and the NH(2)-terminal extension. A 19-amino-acid internal portion of proRIP2 has little similarity to the proRIP1 sequence except that both are very rich in acidic residues. RIP activity assays revealed that Rip3:2 encodes a polypeptide that acquires RNA-specific N-glycosidase activity after proteolytic cleavage. Accumulation as inactive proenzymes may therefore be a general feature of maize RIPs. Differential regulation of the two RIP genes suggests that the corresponding proteins may be involved in defence-related functions with one being regulated developmentally and the other being responsive to an environmental stimulus.

Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress.

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.