Faye M. Rosin

RNA Interference Silencing of Chalcone Synthase, the First Step in the Flavonoid Biosynthesis Pathway, Leads to Parthenocarpic Tomato Fruits

Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed.

Overexpression of a knotted-like homeobox gene of potato alters vegetative development by decreasing gibberellin accumulation.

Potato (Solanum tuberosum) homeobox 1 (POTH1) is a class I homeobox gene isolated from an early-stage tuber cDNA library. The RNA expression pattern ofPOTH1, unlike that of most other class Iknotted-like homeobox genes, is widespread in the cells of both indeterminate and differentiated tissues. Using in situ hybridization, POTH1 transcripts were detected in meristematic cells, leaf primordia, and the vascular procambium of the young stem. Overexpression of POTH1 produced dwarf plants with altered leaf morphology. Leaves were reduced in size and displayed a “mouse-ear” phenotype. The mid-vein was less prominent, resulting in a palmate venation pattern. The overall plant height of overexpression lines was reduced due to a decrease in internode length. Levels of intermediates in the gibberellin (GA) biosynthetic pathway were altered, and the bioactive GA, GA1, was reduced by one-half in sense mutants. Accumulation of mRNA for GA 20-oxidase1, a key biosynthetic enzyme, decreased in overexpression lines. In vitro tuberization was enhanced under both short- and long-day photoperiods in several POTH1 overexpression lines. Sense lines produced more tubers at a faster rate than controls. These results imply that POTH1 mediates the development of potato by acting as a negative regulator of GA biosynthesis.

Interacting Transcription Factors from the Three-Amino Acid Loop Extension Superclass Regulate Tuber Formation

Using the yeast (Saccharomyces cerevisiae) two-hybrid system and a potato (Solanum tuberosum) KNOX protein, designated POTH1, as bait, we have identified seven distinct interacting proteins from a stolon library of potato. All seven cDNAs are members of the BEL1-like family of transcription factors. Among these proteins, there are at least four regions of high sequence conservation including the homeodomain, the proline-tyrosine-proline three-amino acid loop extension, the SKY box, and a 120-amino acid region upstream from the homeodomain. Through deletion analysis, we identified a protein-binding domain present in the carboxy end of the KNOX domain of POTH1. The protein-binding domain in the BEL1 protein is located in the amino-terminal one-half of the 120-residue conserved region of the BELs. RNA-blot analysis showed differential patterns of RNA accumulation for the BELs in various potato organs. The level of StBEL5 mRNA increased in response to a short-day photoperiod in both leaves and stolons. Similar to sense mutants of POTH1, transgenic lines that overexpressed StBEL5 exhibited enhanced tuber formation even under noninductive conditions. Unlike POTH1 sense lines, however, these BEL lines did not exhibit the extreme leaf and stem morphology characteristic of KNOX overexpressers and displayed a more rapid rate of growth than control plants. Both StBEL5 and POTH1 sense lines exhibited an increase in cytokinin levels in shoot tips. StBEL5 lines also exhibited a decrease in the levels of GA 20-oxidase1 mRNA in stolon tips from long-day plants. Our results demonstrate an interaction between KNOX and BEL1-like transcription factors of potato that may potentially regulate processes of development.

Suppression of a Vegetative MADS Box Gene of Potato Activates Axillary Meristem Development

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an early stage tuber cDNA library. The RNA ofPOTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum), accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, and newly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apical dominance accompanied by a compact growth habit and a reduction in leaf size. Suppression lines produced truncated shoot clusters from stem buds and, in a model system, exhibited enhanced axillary bud growth instead of producing a tuber. This enhanced axillary bud growth was not the result of increased axillary bud formation. Tuber yields were reduced and rooting of cuttings was strongly inhibited inPOTM1 suppression lines. Both starch accumulation and the activation of cell division occurred in specific regions of the vegetative meristems of the POTM1 transgenic lines. Cytokinin levels in axillary buds of a transgenic suppression line increased 2- to 3-fold. These results imply that POTM1mediates the control of axillary bud development by regulating cell growth in vegetative meristems.

Old dogs, new tricks: regulatory evolution in conserved genetic modules leads to novel morphologies in plants.

The importance of regulatory evolution to the diversification of plant morphology is well recognized. Some of the best-understood examples also involve gene duplication and co-option of deeply conserved genetic modules. These instances underscore the important role of gene duplication events, which are associated with regulatory sub- and neofunctionalization. In particular, we discuss the relationship between regulatory evolution following gene duplication and the evolution of floral novelty. We also consider the repeated co-option of TCP gene family members to promote aspects of floral symmetry and the KNOX/ARP meristem genetic module to control compound leaf development. Both of these patterns of genetic convergence involve modifications of an ancestral regulatory network to create novel expression domains. Overall, such examples highlight the interdependence of the three processes--regulatory evolution, gene duplication and co-option--within the context of plant developmental evolution.

Molecular controls of tuberization

Tuber formation in potatoes (Solanum tuberosum L.) is a complex developmental process involving a number of important biological systems. Under conditions of a short-day photoperiod and cool temperature, a transmissible signal is activated that initiates cell division and expansion and a change in the orientation of cell growth in the subapical region of the stolon tip. In this signal transduction pathway, perception of the appropriate environmental cues occurs in leaves and is mediated by phytochrome and gibberellins (GA). Phytohormones also play a prominent role in regulating the morphological events of tuberization activated in the stolon apex. GA, cytokinins, and jasmonate-like compounds have all been implicated in regulating tuber development. High levels of GA are correlated with the inhibition of tuberization, whereas low levels are associated with induction. Transcription factors are proteins that bind to DNA to regulate gene activity and, in some cases, to mediate hormone levels. Several of these DNA-binding proteins are involved in regulating plant growth and meristem development in potato, including tuber formation. One type, designated POTM1, regulates cytokinin levels in potato meristems and controls branching of axillary shoots. Two other types that physically interact, the BEL and KNOX proteins, mediate vegetative development. Transgenic plants that overexpressed BEL and KNOX proteins exhibited enhanced tuber formation even under long-day conditions. KNOX overexpressers exhibited abnormal leaf architecture and dwarfism. These transgenic lines exhibited a decrease in the levels of GA and an increase in cytokinin levels. In addition, the BEL transgenic lines grew more rapidly than wild-type plants. Our results indicate that DNA-binding proteins of potato mediate tuberization by enhancing or repressing the activity of specific target genes.ResumenLa formación de tubérculos en papa (Solanum tuberosum L.) es un proceso complejo de desarrollo que compromete diferentes sistemas biológicos importantes. Bajo condiciones de foto período corto y temperatura fría, se activa una señal transmisible en la región subapical del estolón que inicia la división celular y la expansión y cambio de orientatión del crecimiento de las células. En esta via de transducción se realiza la percepción de las señales medio-ambientales apropiadas en las hojas, lo cual se obtiene por mediación del fitocromo y las giberelinas (GA). Las fitohormonas también juegan un rol prominente, regulando los eventos morfológicos de tuberización activados en el ápice del estolón. Las GA, citoquininas y compuestos como el jasmonato han sido implicados en la regulacion del desarrollo del tubérculo. Los niveles altos de GA están correlacionados con la inhibición de la tuberización, mientras que los niveles bajos están asociados con la inducción. Los factores de trascripción son proteínas que se unen al ADN para regular la actividad de los genes y en algunos casos, para regular los niveles hormonales. Varias de estas ligaduras proteicas del ADN están involucradas en la regulacion del crecimiento de la planta y el desarrollo de los meristemos en papa, incluyendo la formación de tubérculos. Un tipo denominado POTM1, regula los niveles de citoquinina en los meristemos de papa y controla la ramificación de los brotes axilares. Otros dos tipos que interactúan físicamente son las proteínas BEL y KNOX que intervienen en el desarrollo vegetativo. Las plantas transgénicas con sobre-producción de proteínas BEL y KNOX incrementaron la formacion de tubérculos aún en condiciones de día largo. Las plantas que sobreexpresaron el KNOX exhibieron una arquitectura anormal en las hojas y enanismo. Estas líneas transgénicas mostraron una disminución en los niveles de AG y un aumento en los niveles de citoquininas. Además, las líneas transgénicas BEL crecieron con mayor rapidez que las plantas del tipo silvestre. Nuestros resultados indican que las proteínas que ligan el ADN de papa, intervienen en la tuberización, aumentando o disminuyendo la actividad de los genes objetivo específicos.

Expression patterns of a putative homolog of AGAMOUS, STAG1, from strawberry

MADS box genes function to regulate vegetative, floral, and fruit development in plants. Here we characterize the expression pattern of a MADS box gene from strawberry (Fragaria � /ananassa ), designated STAG1 . Sequence analysis revealed that STAG1 shared 68 � /91% amino acid sequence identity to AGAMOUS homologs from a variety of plant species. STAG1 transcripts were detected in stamens, carpels, and developing fruit. In situ hybridization revealed that STAG1 mRNA expression was restricted to the endothelium and the vascular bundles connecting the achenes to the inner part of the receptacle and was not evident in the receptacle of the fruit. Analysis of the expression of a GUS marker gene driven by the STAG1 promoter showed that during floral development, STAG1 was active in stamens, the base of the receptacle and the petals, and in the central pith and vascular tissue. During the ripening stage of fruit development, STAG1 activity was detected in achenes, pith cells, and cortical cells. Sequence analysis and expression patterns indicate that STAG1 is an AGAMOUS homolog of strawberry. # 2003 Elsevier Ireland Ltd. All rights reserved.

Genomic level views of novel floral organ morphology

Across the angiosperms there are many examples of independently derived, novel floral organs. The presence of such structures results in five or more distinct floral organ identities, which is difficult to reconcile with the canonical ABC model. In the emerging model system Aquilegia there are five differentiated floral organ types: petaloid sepals, spurred petals, stamens, staminodia and carpels. This morphology would seem to require modifications of the ABC model, namely the capacity to specify two types of petaloid organs as well as a fifth organ identity. Detailed expression studies of the three APETALA3 (AP3) paralogs and one PISTILLATA (PI) homolog in Aquilegia demonstrate that each organ type expresses a specific combination of genes. Furthermore, RNAi-mediated knock-down of B homolog function has found that these loci are essential to staminodium, stamen and petal identity, but play only subtle developmental roles in the petaloid sepals. Our findings show that pre-existing floral organ identity programs can be partitioned and modified to produce additional organ types. As an extension of this work, we are using RNAi to knock down the function of each AP3 paralog independently, which has revealed evidence for both suband neofunctionalization. To complement our candidate gene approach, we are using Illumina-based sequencing of homeotic mutants as well as laser microdissected samples to gain greater insight into the genetic basis of identity and developmental elaboration in the novel floral organs of Aquilegia.