Small RNA-regulated networks and the evolution of novel structures in plants.

The evolution of plants on land has produced a great diversity of organs, tissues, and cell types. Many of the genes identified as having a role in the development of such structures in flowering plants are conserved across all land plants, including in clades that diverged before the evolution of the structure in question. This suggests that novel organs commonly evolve via the cooption of existing developmental gene regulatory networks (GRNs). Although numerous examples of such cooptions have been identified, little is known about why those specific GRNs have been coopted. In this review, we discuss the properties of GRNs that may favor their cooption, as well as the mechanisms by which this can occur, in the context of plant developmental evolution. We especially focus on small RNA (sRNA)-regulated and auxin-signaling GRNs as intriguing models of regulatory network recruitment.

Genetic and epigenetic regulation of stem cell homeostasis in plants.

Plants generate new organs through the activity of small populations of stem cells present in specialized niches called meristems. Stem cell homeostasis is attained by dynamic regulatory networks involving transcriptional regulators, hormones, and other intercellular signals that specify cell fate and convey positional information to the apical stem cells and the organizing center located immediately below. The balance between stem cell maintenance within the shoot apical meristem (SAM) and differentiation of cells that are displaced from the niche to form new organs involves the epigenetic silencing of stem cell regulatory genes. Recent advances have identified highly conserved chromatin remodeling factors as epigenetic regulators of stem cell fate that confer plasticity in plant development and ensure the stable inheritance of repressed expression states during organogenesis. These advances reveal that common mechanisms contribute to stem cell homeostasis in plants and animals.

Organ polarity in plants is specified through the opposing activity of two distinct small regulatory RNAs.

Small RNAs and their targets form complex regulatory networks that control cellular and developmental processes in multicellular organisms. In plants, dorsoventral (adaxial/abaxial) patterning provides a unique example of a developmental process in which early patterning decisions are determined by small RNAs. A gradient of microRNA166 on the abaxial/ventral side of the incipient leaf restricts the expression of adaxial/dorsal determinants. Another class of small RNAs, the TAS3-derivated trans-acting short-interfering RNAs (ta-siRNAs), are expressed adaxially and repress the activity of abaxial factors. Loss of maize leafbladeless1 (lbl1) function, a key component of the ta-siRNA biogenesis pathway, leads to misexpression of miR166 throughout the initiating leaf, implicating ta-siRNAs in the spatiotemporal regulation of miR166. The spatial restriction of tasiRNA biogenesis components suggests that this pathway may act non-cell-autonomously in the meristem and possibly contributes to the classic meristem-borne adaxializing Sussex signal. Here, we discuss the key participants in adaxial/abaxial patterning and point out the intriguing possibility that organ polarity in plants is established by the opposing action of specific ta-siRNAs and miRNAs.

ROUGH SHEATH2: a Myb protein that represses knox homeobox genes in maize lateral organ primordia.

The regulation of members of the knotted1-like homeobox (knox) gene family is required for the normal initiation and development of lateral organs. The maize rough sheath2 (rs2) gene, which encodes a Myb-domain protein, is expressed in lateral organ primordia and their initials. Mutations in the rs2 gene permit ectopic expression of knox genes in leaf and floral primordia, causing a variety of developmental defects. Ectopic KNOX protein accumulation in rs2 mutants occurs in a subset of the normal rs2-expressing cells. This variegated accumulation of KNOX proteins in rs2 mutants suggests that rs2 represses knox expression through epigenetic means.

Interchromosomal recombination in Zea mays.

A new allele of the 27-kD zein locus in maize has been generated by interchromosomal recombination between chromosomes of two different inbred lines. A continuous patch of at least 11,817 bp of inbred W64A, containing the previously characterized Ra allele of the 27-kD zein gene, has been inserted into the genome of A188 by a single crossover. While both junction sequences are conserved, sequences of the two homologs between these junctions differ considerably. W64A contains the 7313-bp-long retrotransposon, Zeon-1. A188 contains a second copy of the 27-kD zein gene and a 2-kb repetitive element. Therefore, recombination results in a 7.3-kb insertion and a 14-kb deletion compared to the original S+A188 allele. If nonpairing sequences are looped out, 206 single base changes, frequently clustered, are present. The structure of this allele may explain how a recently discovered example of somatic recombination occurred in an A188/W64A hybrid. This would indicate that despite these sequence differences, pairing between these alleles could occur early during plant development. Therefore, such a somatically derived chimeric chromosome can also be heritable and give rise to new alleles.

Leafbladeless1 is required for dorsoventrality of lateral organs in maize.

The maize leafbladeless1 (lbl1) mutant displays a variety of leaf and plant phenotypes. The most extreme manifestation in the leaf is the formation of radially symmetric, abaxialized leaves due to a complete loss of adaxial cell types. Less severe phenotypes, resulting from a partial loss of adaxial cell identity, include the formation of ectopic laminae at the boundary between abaxialized, mutant sectors on the adaxial leaf surface and the bifurcation of leaves. Ectopic laminae and bifurcations arise early in leaf development and result in an altered patterning of the leaf along the proximodistal axis, or in complete duplication of the developing organ. Leaf-like lateral organs of the inflorescences and flowers show similar phenotypes. These observations suggest that Lbl1 is required for the specification of adaxial cell identity within leaves and leaf-like lateral organs. Lbl1 is also required for the lateral propagation of leaf founder cell recruitment, and plays a direct or indirect role in the downregulation of the homeobox gene, knotted1, during leaf development. Our results suggest that adaxial/abaxial asymmetry of lateral organs is specified in the shoot apical meristem, and that formation of this axis is essential for marginal, lateral growth and for the specification of points of proximodistal growth. Parallels between early patterning events during lateral organ development in plants and animals are discussed.

Dynamic genome organization and gene evolution by positive selection in geminivirus (Geminiviridae).

Geminiviruses (Geminiviridae) are a diverse group of plant viruses differing from other known plant viruses in possessing circular, single-stranded DNA. Current classification divides the family into three subgroups, defined in part by genome organization, insect vector, and plant host range. Previous phylogenetic assessments of geminiviruses have used DNA and/or amino acid sequences from the replication-associated and coat protein genes and have relied predominantly on distance analyses. We used amino acid and DNA sequence data from the replication-associated and coat protein genes from 22 geminivirus types in distance and parsimony analyses. Although the results of our analyses largely agree with those reported previously, we could not always predict viral relationships based on genome organization, plant host, or insect vector. Loss of correlation of these traits with phylogeny is likely due to improved sampling of geminivirus types. Unrooted parsimony trees suggest multiple independent origins for the monopartite genome. genome organization is therefore a dynamic character. Estimates of nonsynonymous and synonymous nucleotide substitutions for extant and inferred ancestral sequences were used to evaluate hypotheses that the replication-associated and coat protein sequences evolve to accommodate plant host and insect vector specificities, respectively. Results suggest that plant host specificity does not solely direct replication-associated protein-evolution but that coat protein sequence does evolve in response to insect vector specificity. Genome organization and, possibly, plant host specificity are not reliable taxonomic characters.

Region-specific cis- and trans-acting factors contribute to genetic variability in meiotic recombination in maize.

Understanding the genetic basis for variability in recombination rates is important for general genetic studies and plant-breeding efforts. Earlier studies had suggested increased recombination frequencies in particular F2 populations derived from the maize inbred A188. A detailed phenotypic and molecular analysis was undertaken to extend these observations and dissect the responsible factors. A heritable increase in recombination in the sh1-bz1 interval was observed in these populations. A factor causing an approximate twofold increase mapped to the A188 sh1-Bz1 region, behaved as a dominant, cis-acting factor, affected recombination equally in male and female sporogenesis and did not reduce the well-studied complete interference in the adjacent bz1-wx interval. This factor also did not increase recombination frequencies in the c1-sh1 and bz1-wx intervals, demonstrating independent control of recombination in adjacent intervals. Additional phenotypic analysis of recombination in the c1-sh1 and bz1-wx intervals and RFLP analysis of recombination along chromosomes 7 and 5 suggested that heritable factors controlling recombination in these intervals act largely independently and in trans. Our results show that recombination in these populations, and possibly maize in general, is controlled by both cis- and trans-acting factors that affect specific chromosomal regions.

Characterization of a meiotic crossover in maize identified by a restriction fragment length polymorphism-based method.

Genetic map lengths do not correlate directly with genome size, suggesting that meiotic recombination is not uniform throughout the genome. Further, the abundance of repeated sequences in plant genomes requires that crossing over is restricted to particular genomic regions. We used a physical mapping approach to identify these regions without the bias introduced by phenotypic selection. This approach is based on the detection of nonparental polymorphisms formed by recombination between polymorphic alleles. In an F2 population of 48 maize plants, we identified a crossover at two of the seven restriction fragment length polymorphism loci tested. Characterization of one recombination event revealed that the crossover mapped within a 534-bp region of perfect homology between the parental alleles embedded in a 2773-bp unique sequence. No transcripts from this region could be detected. Sequences immediately surrounding the crossover site were not detectably methylated, except for an SstI site and at the flanking repetitive sequences were faithfully inherited by the recombinant allele. Our observations suggest that meiotic recombination in maize occurs between perfectly homologous sequences, within unmethylated, nonrepetitive regions of the genome.

Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize.

The 18 kDa high-methionine delta-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of delta-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine delta-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown.

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.

In vitro deletion mapping of the viral strand replication origin of Pseudomonas bacteriophage Pf3.

The origin of viral strand replication of the filamentous bacteriophage Pf3 has been characterized in Escherichia coli by in vitro deletion mapping techniques. The origin region was functionally identified by its ability to convey replicative properties to a recombinant plasmid in a polA host in which the replication origin of the vector plasmid is not functional. The origin of Pf3 viral strand replication is contained within a DNA sequence of 139 bp. This sequence covers almost completely one of the intergenic regions of the Pf3 genome, and it specifies both replication initiation and termination functions. Although no nucleotide sequence homology is present between the Pf3 origin of viral strand replication and that of the E. coli filamentous phages Ff (M13, f1, and fd) and IKe, their map positions and functional properties are very similar.

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.

The pFF plasmids: cassettes utilising CaMV sequences for expression of foreign genes in plants.

A plant expression cassette was constructed using the cauliflower mosaic virus 35S 5' regulatory region with the enhancer duplicated and the 35S polyadenylation signal. Insertion of a polylinker between the transcription initiation and polyadenylation sites allows for easy cloning of genes. To test the usefulness of the cassette chimeric bacterial genes were prepared. The constructs were introduced into Nicotiana tabacum suspension culture cells by the particle bombardment process. Expression of the beta-glucuronidase reporter gene was verified by histochemical staining. Stable kanamycin and hygromycin resistant transgenic lines were obtained after introduction of chimeric genes encoding the enzymes neomycin phosphotransferase and hygromycin B phosphotransferase, respectively. The number of stable transformants was approximately 2% of the cells that transiently expressed the beta-glucuronidase reporter gene.