David R. Smyth

LEAFY controls floral meristem identity in Arabidopsis

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.

TRANSPARENT TESTA GLABRA2, a Trichome and Seed Coat Development Gene of Arabidopsis, Encodes a WRKY Transcription Factor

Mutants of a new gene, TRANSPARENT TESTA GLABRA2 (TTG2), show disruptions to trichome development and to tannin and mucilage production in the seed coat. The gene was tagged by the endogenous transposon Tag1 and shown to encode a WRKY transcription factor. It is the first member of this large, plant-specific family known to control morphogenesis. The functions of all other WRKY genes revealed to date involve responses to pathogen attack, mechanical stress, and senescence. TTG2 is strongly expressed in trichomes throughout their development, in the endothelium of developing seeds (in which tannin is later generated) and subsequently in other layers of the seed coat, and in the atrichoblasts of developing roots. TTG2 acts downstream of the trichome initiation genes TTG1 and GLABROUS1, although trichome expression of TTG2 continues to occur if they are inactivated. Later, TTG2 shares functions with GLABRA2 in controlling trichome outgrowth. In the seed coat, TTG2 expression requires TTG1 function in the production of tannin. Finally, TTG2 also may be involved in specifying atrichoblasts in roots redundantly with other gene(s) but independently of TTG1 and GLABRA2.

An abundant LINE-like element amplified in the genome of Lilium speciosum.

SummaryThe genomes of Lilium species are very large, containing 30–40 million kilobase pairs of DNA. An abundant fragment of 3.5 kb was released by BamHI digestion of genomic DNA of Lilium speciosum. Analysis of 20 genomic clones containing sequences homologous to the fragment showed it to be part of a 4.45 kb dispersed repeat, which was named del2. Sequence analysis of one full element and regions of four others revealed del2 to be a non-LTR (long terminal repeat) retrotransposon. It is flanked by short direct repeats of from 4 to 13 bp and a run of adenines occurs at one end (the proposed 3′ end), 63 by downstream from a polyadenylation signal. A possible RNA polymerase II promoter similar to that found in Drosophila I and F group elements is present internally 30 by downstream from the 5′ end. Two degenerate open reading frames (ORFs) are present, the 5′ ORF containing a gag-related cysteine motif, and the 3′ ORF containing a different cysteine motif also found in most non-LTR retrotransposons. The 3′ ORF also has regions with homology to reverse transcriptase sequences, which are most similar to those in Cin4 of maize, the Ll LINE elements of humans and mice and the R2 ribosomal DNA inserts of insects. The majority of del2 elements occur as the full 4.45 kb element. They account for an estimated 4 % of the L. speciosum genome and are present in approximately 250 000 copies. del2-related sequences were also detected in 12 other monocot species. del2 is the most abundant non-LTR retrotransposon identified so far and reveals that LINE-like elements have been greatly amplified in some plant genomes just as they have in mammals.

PETAL LOSS, a trihelix transcription factor gene, regulates perianth architecture in the Arabidopsis flower.

Perianth development is specifically disrupted in mutants of the PETAL LOSS (PTL) gene, particularly petal initiation and orientation. We have cloned PTL and show that it encodes a plant-specific trihelix transcription factor, one of a family previously known only as regulators of light-controlled genes. PTL transcripts were detected in the early-developing flower, in four zones between the initiating sepals and in their developing margins. Strong misexpression of PTL in a range of tissues universally results in inhibition of growth, indicating that its normal role is to suppress growth between initiating sepals, ensuring that they remain separate. Consistent with this, sepals are sometimes fused in ptl single mutants, but much more frequently in double mutants with either of the organ boundary genes cup-shaped cotyledon1 or 2. Expression of PTL within the newly arising sepals is apparently prevented by the PINOID auxin-response gene. Surprisingly, PTL expression could not be detected in petals during the early stages of their development, so petal defects associated with PTL loss of function may be indirect, perhaps involving disruption to signalling processes caused by overgrowth in the region. PTL-driven reporter gene expression was also detected at later stages in the margins of expanding sepals, petals and stamens, and in the leaf margins; thus, PTL may redundantly dampen lateral outgrowth of these organs, helping define their final shape.

INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis

Mobile signals provided by hormones and morphogens are essential to organize multicellular structures. This article demonstrates that the joint activity of two bHLH transcription factors is required at two separate stages during Arabidopsis gynoecium and fruit development. In both instances, these factors mediate their function by ensuring appropriate distribution of the plant hormone auxin. Structural organization of organs in multicellular organisms occurs through intricate patterning mechanisms that often involve complex interactions between transcription factors in regulatory networks. For example, INDEHISCENT (IND), a basic helix-loop-helix (bHLH) transcription factor, specifies formation of the narrow stripes of valve margin tissue, where Arabidopsis thaliana fruits open on maturity. Another bHLH transcription factor, SPATULA (SPT), is required for reproductive tissue development from carpel margins in the Arabidopsis gynoecium before fertilization. Previous studies have therefore assigned the function of SPT to early gynoecium stages and IND to later fruit stages of reproductive development. Here we report that these two transcription factors interact genetically and via protein–protein contact to mediate both gynoecium development and fruit opening. We show that IND directly and positively regulates the expression of SPT, and that spt mutants have partial defects in valve margin formation. Careful analysis of ind mutant gynoecia revealed slight defects in apical tissue formation, and combining mutations in IND and SPT dramatically enhanced both single-mutant phenotypes. Our data show that SPT and IND at least partially mediate their joint functions in gynoecium and fruit development by controlling auxin distribution and suggest that this occurs through cooperative binding to regulatory sequences in downstream target genes.

CRABS CLAW and SPATULA Genes Regulate Growth and Pattern Formation during Gynoecium Development in Arabidopsis thaliana

Development of the gynoecium of Arabidopsis is disrupted in mutants of the regulatory genes SPATULA (SPT; a basic helix‐loop‐helix family member) and CRABS CLAW (CRC; a YABBY family gene). We have defined the disruptions in detail, plotting their time course during gynoecial development, mapping disruptions to xylem lignification, and testing their effects on fertilization. In spt mutants, defects were first seen soon after the gynoecial tube started to elongate. Medial regions where carpels adjoin grew slower than in wild type and were often unfused later at the apex. Development of the septum was severely disrupted, and extracellular matrix‐secreting transmitting tract was not seen in null mutant lines. Even so, some pollination was observed. The amount of stylar and stigmatic tissue was also reduced, and vascular development in medial and stylar regions was disrupted. SPT apparently plays a role in promoting the development of all specialized tissues from carpel margins. In crc mutants, defects were very different. They were seen from the inception of gynoecial development and characterized by additional cells arising across the width of the gynoecium but fewer cells in the longitudinal dimension. In addition, cells were larger on average. All cell types arose in crc mutant gynoecia, but they seemed to differentiate earlier. Mutant gynoecia were always unfused in apical regions, and the medial vasculature was again disrupted. CRC may normally restrain lateral cell division but promote longitudinal division, ensuring the gynoecium adopts an elongated linear form. In crc spt double mutants, nonadditive disruptions were present, with the carpels even shorter, much less fused, and lacking stylar xylem elements. These interactions may be secondary, however, as the expression patterns of the two genes do not overlap. Disruptions in both spt and crc mutants match closely the time and place of expression of the wild‐type genes, indicating that each acts cell autonomously. We have integrated these findings with those of other known regulatory genes to propose a general model of growth and pattern formation in the developing gynoecium. In an initial “neogenic” phase, lateral and medial regions are defined. Meristematic genes (including SPT) are active in the medial zone, maintaining its pluripotent potential, while growth and polarity genes (including CRC) are active in the lateral zones, providing the valve initials with the “competence” to support medial tissue. Later, in a “determination and differentiation” phase, the medial zone is genetically divided into differentiating subregions, with septum and placentae developing internally and the replum developing externally. At the same time, the lateral regions mature into the tissues of the ovary wall.

A survey of C-band patterns in chromosomes ofLilium (Liliaceae)

C-band patterns are described for 20Lilium spp. distributed across six sections. All species have a similar basic karyotype (n = 12) but C-bands differ markedly between them. The patterns are characterized by a dispersed scattering of thin intercalary bands as well as centric and NOR bands. Only one species,L. canadense, shows a clear equilocal pattern with intercalary C-bands occurring proximally in all of the longer chromosome arms. Comparing species, similar patterns are revealed forL. regale andL. sulphureum, forL. formosanum andL. longiflorum (all in sect.Leucolirion) and to a lesser extent forL. hansonii, L. martagon, andL. tsingtauense (sect.Martagon). The pattern forL. henryi (previously classed in sect.Sinomartagon) matches those ofL. regale andL. sulphureum quite well and its transfer to sect.Leucolirion is proposed. This is consistent with results from interspecies hybrids betweenL. henryi andL. regale (and related species) which are reportedly fertile. No other clear similarities in C-band patterns were seen across species. It seems that C-band patterns change rapidly inLilium and hence their usefulness in classification will be restricted to identifying closely related species.

The trihelix family of transcription factors – light, stress and development

GT factors are the founding members of the trihelix transcription factor family. They bind GT elements in light regulated genes, and their nature was uncovered in a burst of activity in the 1990s. Study of the trihelix family then slowed. However, interest is now re-awakening. Genomic studies have revealed 30 members of this family in Arabidopsis and 31 in rice, falling into five clades. Newly discovered functions involve responses to salt and pathogen stresses, the development of perianth organs, trichomes, stomata and the seed abscission layer, and the regulation of late embryogenesis. Thus the time is ripe for a review of the genomic and functional information now emerging for this neglected family.

The ABC model of flower development: then and now

In 1991, we published a paper in Development that proposed the ABC model of flower development, an early contribution to the genetic analysis of development in plants. In this, we used a series of homeotic mutants, and double and triple mutants, to establish a predictive model of organ specification in developing flowers. This model has served as the basis for much subsequent work, especially towards understanding seed plant evolution. Here, we discuss several aspects of this story, that could be a much longer one. One surprising conclusion is that materials and methods that might have led to similar work, and to the same model, were available 100 years before our experiments, belying the belief that progress in biology necessarily comes from improvements in methods, rather than in concepts.

Dispersed repeats in plant genomes

The amount of DNA in an unreplicated haploid cell (the C value) is relatively constant within a species. However in higher plants it is particularly variable between species, ranging over nearly three orders of magnitude (Bennett and Smith 1976). The lowest amount recorded to date is in the ephemeral crucifer, Arabidopsis thaliana. Results from reannealing experiments originally suggested that this species has around 70000 kb per genome (Leutwiler et al. 1984), although recent data from genomic libraries have put it closer to 100000 kb. At the top of the scale lie various monocot species. For example, the true lilies (Lilium species) have around 3040 million kb per genome, while the record belongs to Fritillaria species, also bulbous monocots but with about twice the level of their Lilium relatives. Thus plant gehomes can range in size from 100000 to nearly 100000000 kb! And yet the structural and developmental complexity of pIant species with the Iowest amounts of DNA per cell is not fundamentally different from those with the highest. The number of different genes required to