Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro.

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.

Genome-wide transposon tagging reveals location-dependent effects on transcription and chromatin organization in Arabidopsis.

The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.

Moonlighting vacuolar protease: multiple jobs for a busy protein.

In this Genomics Era with a wealth of annotated sequence data, it is easy to pigeonhole a protein into a particular function. However, Noa Matarasso et al. recently found a vacuolar protease that can also function as a transcription factor. This work illustrates that a protein can serve multiple roles in a cell, raising intriguing questions as to the extent that genomic information can be deciphered de novo.

Size and number of tandem repeat arrays can determine somatic homologous pairing of transgene loci mediated by epigenetic modifications in Arabidopsis thaliana nuclei.

The chromosomal arrangement of different transgenic repeat arrays inserted at various chromosomal positions was tested by FISH in Arabidopsis 2C leaf and root nuclei. Large lacO ( approximately 10 kb) but not tetO (4.8 kb) or small lacO ( approximately 2 kb) arrays were, in general, more often spatially associated with heterochromatic chromocenters (CC) than flanking regions (that either overlap the array insert position or are between 5 and 163 kb apart from the insert site). Allelic and ectopic pairing frequencies of lacO arrays were significantly increased only in nuclei of lines with two large lacO arrays inserted at different positions on the same chromosome arm. Within the same lines, root nuclei showed a significantly lower increase of pairing frequencies at the insert position compared to leaf nuclei but still a higher frequency than in the wild-type situation. Thus, the frequencies of homologous pairing and association with heterochromatin of transgenic repeats may differ with the construct, the chromosomal insertion position, the cell type and with the number and repetitiveness of inserts. Strong CpG methylation is correlated with a high frequency of homologous pairing at large repeat array loci in somatic cells but has no impact on their association with CCs. These results show that single low-copy arrays apparently do not alter interphase chromatin architecture and are more suitable for chromatin tagging than multiple high copy arrays.

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.

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 of POTM1 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 in POTM1 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 POTM1 mediates the control of axillary bud development by regulating cell growth in vegetative meristems.

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.

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 of POTH1, unlike that of most other class I knotted-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, GA(1), 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.