Peter M. Rogowsky

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family

Abstract. Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (omatic mbryogenesis eceptor-like inase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Epidermis: the formation and functions of a fundamental plant tissue

Epidermis differentiation and maintenance are essential for plant survival. Constant cross-talk between epidermal cells and their immediate environment is at the heart of epidermal cell fate, and regulates epidermis-specific transcription factors. These factors in turn direct epidermal differentiation involving a whole array of epidermis-specific pathways including specialized lipid metabolism necessary to build the protective cuticle layer. An intact epidermis is crucial for certain key processes in plant development, shoot growth and plant defence. Here, we discuss the control of epidermal cell fate and the function of the epidermal cell layer in the light of recent advances in the field.

Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation

We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.

PPR2263, a DYW-Subgroup Pentatricopeptide Repeat Protein, Is Required for Mitochondrial nad5 and cob Transcript Editing, Mitochondrion Biogenesis, and Maize Growth

The work identifies maize PPR2263 and Arabidopsis thaliana MEF29 as orthologous mitochondrial RNA editing proteins, the first such orthologs shown to share target sites between a monocot and a dicot. In maize, the loss of editing of the cob transcript by PPR2263 causes the loss of a protein complex in the mitochondrial respiratory chain and ultimately slow growth of mutant plants. RNA editing plays an important role in organelle gene expression in various organisms, including flowering plants, changing the nucleotide information at precise sites. Here, we present evidence that the maize (Zea mays) nuclear gene Pentatricopeptide repeat 2263 (PPR2263) encoding a DYW domain–containing PPR protein is required for RNA editing in the mitochondrial NADH dehydrogenase5 (nad5) and cytochrome b (cob) transcripts at the nad5-1550 and cob-908 sites, respectively. Its putative ortholog, MITOCHONDRIAL EDITING FACTOR29, fulfills the same role in Arabidopsis thaliana. Both the maize and the Arabidopsis proteins show preferential localization to mitochondria but are also detected in chloroplasts. In maize, the corresponding ppr2263 mutation causes growth defects in kernels and seedlings. Embryo and endosperm growth are reduced, leading to the production of small but viable kernels. Mutant plants have narrower and shorter leaves, exhibit a strong delay in flowering time, and generally do not reach sexual maturity. Whereas mutant chloroplasts do not have major defects, mutant mitochondria lack complex III and are characterized by a compromised ultrastructure, increased transcript levels, and the induction of alternative oxidase. The results suggest that mitochondrial RNA editing at the cob-908 site is necessary for mitochondrion biogenesis, cell division, and plant growth in maize.

Nucleotide diversity of the ZmPox3 maize peroxidase gene: relationships between a MITE insertion in exon 2 and variation in forage maize digestibility.

BackgroundPolymorphisms were investigated within the ZmPox3 maize peroxidase gene, possibly involved in lignin biosynthesis because of its colocalization with a cluster of QTL related to lignin content and cell wall digestibility. The purpose of this study was to identify, on the basis of 37 maize lines chosen for their varying degrees of cell wall digestibility and representative of temperate regions germplasm, ZmPox3 haplotypes or individual polymorphisms possibly associated with digestibility.ResultsNumerous haplotypes with high diversity were identified. Frequency of nucleotide changes was high with on average one SNP every 57 bp. Nucleotide diversity was not equally distributed among site categories: the estimated π was on average eight times higher for silent sites than for non-synonymous sites. Numerous sites were in linkage disequilibrium that decayed with increasing physical distance. A zmPox3 mutant allele, carrying an insertion of a transposable element in the second exon, was found in lines derived from the early flint inbred line, F7. This element possesses many structural features of miniature inverted-repeat transposable elements (MITE). The mutant allele encodes a truncated protein lacking important functional sites. An ANOVA performed with a subset of 31 maize lines indicated that the transposable element was significantly associated with cell wall digestibility. This association was confirmed using an additional set of 25 flint lines related to F7. Moreover, RT-PCR experiments revealed a decreased amount of corresponding mRNA in plants with the MITE insertion.ConclusionThese results showed that ZmPox3 could possibly be involved in monolignol polymerisation, and that a deficiency in ZmPox3 peroxidase activity seemingly has a negative effect on cell wall digestibility. Also, genetic diversity analyses of ZmPox3 indicated that this peroxidase could be a relevant target for grass digestibility improvement using specific allele introgressions.

The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize.

Among the genes controlling the differentiation and maintenance of epidermal cell fate are members of the HD-ZIP IV class family of plant-specific transcription factors, most of which are specifically expressed in the epidermis of tissues. Here, we report the functional analysis of the maize HD-ZIP IV gene OCL4 (outer cell layer 4) via the phenotypic analysis of two insertional mutants, and of OCL4-RNAi transgenic plants. In all three materials, the macrohairs, one of the three types of trichomes present on adult maize leaf blades, developed ectopically at the margin of juvenile and adult leaves. Consistent with this phenotype, OCL4 is expressed in the epidermis of the leaf blade, with a maximum at the margin of young leaf primordia. Expression of OCL4 in the model plant Arabidopsis under the control of the GLABRA2 (GL2) promoter, a member of the Arabidopsis HD-ZIP IV family involved in trichome differentiation, did not complement the gl2-1 mutant, but instead aggravated its phenotype. The construct also caused a glabrous appearance of rosette leaves in transformed control plants of the Ler ecotype, suggesting that OCL4 inhibits trichome development both in maize and Arabidopsis. Furthermore, insertional mutants showed a partial male sterility that is likely to result from the presence of an extra subepidermal cell layer with endothecium characteristics in the anther wall. Interestingly, the epidermis-specific OCL4 expression in immature anthers was restricted to the region of the anther locule where the extra cell layer differentiated. Taken together these results suggest that OCL4 inhibits trichome development and influences division and/or differentiation of the anther cell wall.

Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis

Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

SummaryDNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72–73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two clements. A TnpR− derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.