Heidi F. Kaeppler

Comparative Analysis of SET Domain Proteins in Maize and Arabidopsis Reveals Multiple Duplications Preceding the Divergence of Monocots and Dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

Transgene-induced RNA interference as a tool for plant functional genomics

RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.

Sequence Relationships, Conserved Domains, and Expression Patterns for Maize Homologs of the Polycomb Group Genes E(z), esc, and E(Pc)

Polycomb group (PcG) proteins play an important role in developmental and epigenetic regulation of gene expression in fruit fly (Drosophila melanogaster) and mammals. Recent evidence has shown that Arabidopsis homologs of PcG proteins are also important for the regulation of plant development. The objective of this study was to characterize the PcG homologs in maize (Zea mays). The 11 cloned PcG proteins from fruit fly and the Enhancer of zeste[E(z)], extra sex combs(esc), and Enhancer of Polycomb[E(Pc)] homologs from Arabidopsis were used as queries to perform TBLASTN searches against the public maize expressed sequence tag database and the Pioneer Hi-Bred database. Maize homologs were found for E(z), esc, and E(Pc), but not for Polycomb, pleiohomeotic,Posterior sex combs, Polycomblike,Additional sex combs, Sex combs on midleg, polyhometoic, or multi sex combs. Transcripts of the three maize Enhancer of zeste-like genes, Mez1, Mez2, andMez3, were detected in all tissues tested, and theMez2 transcript is alternatively spliced in a tissue-dependent pattern. Zea mays fertilization independent endosperm1 (ZmFie1) expression was limited to developing embryos and endosperms, whereas ZmFie2expression was found throughout plant development. The conservation ofE(z) and esc homologs across kingdoms indicates that these genes likely play a conserved role in repressing gene expression.

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

SummaryGenetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk.

Transgenic oat plants via visual selection of cells expressing green fluorescent protein

Abstract New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Assessing the Efficiency of RNA Interference for Maize Functional Genomics

A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.

Autophagic Recycling Plays a Central Role in Maize Nitrogen Remobilization

Characterization of maize autophagy through detailed transcriptome studies and phenotypic and 15N-partitioning analyses of atg12 mutants reveal a central role for autophagy in nitrogen remobilization. Autophagy is a primary route for nutrient recycling in plants by which superfluous or damaged cytoplasmic material and organelles are encapsulated and delivered to the vacuole for breakdown. Central to autophagy is a conjugation pathway that attaches AUTOPHAGY-RELATED8 (ATG8) to phosphatidylethanolamine, which then coats emerging autophagic membranes and helps with cargo recruitment, vesicle enclosure, and subsequent vesicle docking with the tonoplast. A key component in ATG8 function is ATG12, which promotes lipidation upon its attachment to ATG5. Here, we fully defined the maize (Zea mays) ATG system transcriptionally and characterized it genetically through atg12 mutants that block ATG8 modification. atg12 plants have compromised autophagic transport as determined by localization of a YFP-ATG8 reporter and its vacuolar cleavage during nitrogen or fixed-carbon starvation. Phenotypic analyses showed that atg12 plants are phenotypically normal and fertile when grown under nutrient-rich conditions. However, when nitrogen-starved, seedling growth is severely arrested, and as the plants mature, they show enhanced leaf senescence and stunted ear development. Nitrogen partitioning studies revealed that remobilization is impaired in atg12 plants, which significantly decreases seed yield and nitrogen-harvest index. Together, our studies demonstrate that autophagy, while nonessential, becomes critical during nitrogen stress and severely impacts maize productivity under suboptimal field conditions.

Interaction of Photoperiod and Vernalization Determines Flowering Time of Brachypodium distachyon

The temperate grass, Brachypodium distachyon, is a useful model for studying gene networks controlling flowering. Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Expression of thaumatin-like permatin PR-5 genes switches from the ovary wall to the aleurone in developing barley and oat seeds.

Permatins are antifungal thaumatin-like proteins (TLPs) of the PR-5 family of pathogenesis-related proteins. They occur in many cereals, but little is known of their expression and roles. Permatin cDNA clones were produced and used to study expression in developing barley and oat seeds. Actin and CDC48 mRNAs declined rapidly following inoculation of barley spikes with Fusarium graminearum. Despite this, permatin mRNA levels remained constant or increased slightly. Studies of permatin gene expression in healthy plants revealed that developing barley and oat seeds accumulate permatin mRNA in an unusual bimodal pattern. Permatin mRNA and protein are highly abundant around the time of pollination and then decrease rapidly to near-zero. A second peak occurs in the doughy stage of development. Antibody and DNA probe hybridization studies showed that expression initially occurs in the ovary wall and then switches to the aleurone and ventral furrow of developing seeds, reaching a peak in the doughy stage. Small amounts of permatin mRNAs also occur in certain vegetative tissues. The barley and oat permatin sequences provided sufficient comparisons between cereal TLPs to suggest that deletions or additions in specific elements could have led to the divergence of leaf- and seed-specific TLPs.

Whole transcriptome profiling of maize during early somatic embryogenesis reveals altered expression of stress factors and embryogenesis-related genes

Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species.