Jan Zethof

A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity

It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida and the fistulata (fis) mutant of Antirrhinum majus, which have similar homeotic phenotypes, are recessive alleles of two homologous miRNA-encoding genes. The BL and FIS genes control the spatial restriction of homeotic class C genes to the inner floral whorls, but their ubiquitous early floral expression patterns are in contradiction with a potential role in patterning C gene expression. We provide genetic evidence for the unexpected function of the MIRFIS and MIRBL genes in the center of the flower and propose a dynamic mechanism underlying their regulatory role. Notably, Arabidopsis thaliana, a more distantly related species, also contains this miRNA module but does not seem to use it to confine early C gene expression to the center of the flower.

Analysis of the Petunia TM6 MADS Box Gene Reveals Functional Divergence within the DEF/AP3 Lineage

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.

Differential recruitment of WOX transcription factors for lateral development and organ fusion in Petunia and Arabidopsis.

Petal fusion in petunia (Petunia × hybrida) results from lateral expansion of the five initially separate petal primordia, forming a ring-like primordium that determines further development. Here, we show that MAEWEST (MAW) and CHORIPETALA SUZANNE (CHSU) are required for petal and carpel fusion, as well as for lateral outgrowth of the leaf blade. Morphological and molecular analysis of maw and maw chsu double mutants suggest that polarity defects along the adaxial/abaxial axis contribute to the observed reduced lateral outgrowth of organ primordia. We show that MAW encodes a member of the WOX (WUSCHEL-related homeobox) transcription factor family and that a partly similar function is redundantly encoded by WOX1 and PRESSED FLOWER (PRS) in Arabidopsis thaliana, indicating a conserved role for MAW/WOX1/PRS genes in regulating lateral organ development. Comparison of petunia maw and Arabidopsis wox1 prs phenotypes suggests differential recruitment of WOX gene function depending on organ type and species. Our comparative data together with previous reports on WOX gene function in different species identify the WOX gene family as highly dynamic and, therefore, an attractive subject for future evo-devo studies.

The petunia AGL6 gene has a SEPALLATA-like function in floral patterning

SEPALLATA (SEP) MADS-box genes are required for the regulation of floral meristem determinacy and the specification of sepals, petals, stamens, carpels and ovules, specifically in angiosperms. The SEP subfamily is closely related to the AGAMOUS LIKE6 (AGL6) and SQUAMOSA (SQUA) subfamilies. So far, of these three groups only AGL6-like genes have been found in extant gymnosperms. AGL6 genes are more similar to SEP than to SQUA genes, both in sequence and in expression pattern. Despite the ancestry and wide distribution of AGL6-like MADS-box genes, not a single loss-of-function mutant exhibiting a clear phenotype has yet been reported; consequently the function of AGL6-like genes has remained elusive. Here, we characterize the Petunia hybrida AGL6 (PhAGL6, formerly called PETUNIA MADS BOX GENE4/pMADS4) gene, and show that it functions redundantly with the SEP genes FLORAL BINDING PROTEIN2 (FBP2) and FBP5 in petal and anther development. Moreover, expression analysis suggests a function for PhAGL6 in ovary and ovule development. The PhAGL6 and FBP2 proteins interact in in vitro experiments overall with the same partners, indicating that the two proteins are biochemically quite similar. It will be interesting to determine the functions of AGL6-like genes of other species, especially those of gymnosperms.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Redefining C and D in the Petunia ABC

The petunia AGAMOUS subfamily of MADS box transcription factors contains two C- and two D-lineage genes. This work shows that the two C-genes redundantly specify stamen and carpel identity, whereas all four genes participate in ovule identity specification and floral meristem termination. These results illustrate that subfunctionalization among homeotic genes can vary considerably between species. According to the ABC(DE) model for flower development, C-genes are required for stamen and carpel development and floral determinacy, and D-genes were proposed to play a unique role in ovule development. Both C- and D-genes belong to the AGAMOUS (AG) subfamily of MADS box transcription factors. We show that the petunia (Petunia hybrida) C-clade genes PETUNIA MADS BOX GENE3 and FLORAL BINDING PROTEIN6 (FBP6) largely overlap in function, both in floral organ identity specification and floral determinacy, unlike the pronounced subfunctionalization observed in Arabidopsis thaliana and snapdragon (Antirrhinum majus). Some specialization has also evolved, since FBP6 plays a unique role in the development of the style and stigma. Furthermore, we show that the D-genes FBP7 and FBP11 are not essential to confer ovule identity. Instead, this function is redundantly shared among all AG members. In turn, the D-genes also participate in floral determinacy. Gain-of-function analyses suggest the presence of a posttranscriptional C-repression mechanism in petunia, most likely not existing in Arabidopsis. Finally, we show that expression maintenance of the paleoAPETALA3-type B-gene TOMATO MADS BOX GENE6 depends on the activity of C-genes. Taken together, this demonstrates considerable variation in the molecular control of floral development between eudicot species.

Generation of a 3D indexed Petunia insertion database for reverse genetics

BLAST searchable databases containing insertion flanking sequences have revolutionized reverse genetics in plant research. The development of such databases has so far been limited to a small number of model species and normally requires extensive labour input. Here we describe a highly efficient and widely applicable method that we adapted to identify unique transposon-flanking genomic sequences in Petunia. The procedure is based on a multi-dimensional pooling strategy for the collection of DNA samples; up to thousands of different templates are amplified from each of the DNA pools separately, and knowledge of their source is safeguarded by the use of pool-specific (sample) identification tags in one of the amplification primers. All products are combined into a single sample that is subsequently used as a template for unidirectional pyrosequencing. Computational analysis of the clustered sequence output allows automatic assignment of sequences to individual DNA sources. We have amplified and analysed transposon-flanking sequences from a Petunia transposon insertion library of 1000 individuals. Using 30 DNA isolations, 70 PCR reactions and two GS20 sequencing runs, we were able to allocate around 10 000 transposon flanking sequences to specific plants in the library. These sequences have been organized in a database that can be BLAST-searched for insertions into genes of interest. As a proof of concept, we have performed an in silico screen for insertions into members of the NAM/NAC transcription factor family. All in silico-predicted transposon insertions into members of this family could be confirmed in planta.

Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen long-fin zebrafish: stronger effects in the resting phase than in the active phase

Zebrafish (Danio rerio Hamilton) are increasingly used as a model to study the effects of chronic stress on brain and behaviour. In rodents, unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than during the resting phase. Here, we applied UCS during the daytime (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3 day protocol where fish learn to avoid swimming from a white to a black compartment where they will receive a 3 V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon were quantified. Following 14 days of UCS during the day, inhibitory avoidance learning decreased (lower latencies on days 2 and 3); minor effects were found following 7 days of UCS. Following 7 nights of UCS, inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared with controls (100%) from 7 days of UCS (139%), to 14 days of UCS (174%) to 7 nights of UCS (231%), suggestive of an increasing stress load. Only in the 7 nights of UCS group did expression levels of corticoid receptor genes (mr, grα, grβ) and of bdnf increase. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and as being indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress axis-related genes, especially grβ; in mammals this receptor has been implicated in glucocorticoid resistance and depression.

Knockdown of monocarboxylate transporter 8 (mct8) disturbs brain development and locomotion in zebrafish.

Allan-Herndon-Dudley syndrome (AHDS) is an inherited disorder of brain development characterized by severe psychomotor retardation. This X-linked disease is caused by mutations in the monocarboxylate transporter 8 (MCT8), an important thyroid hormone transporter in brain neurons. MCT8-knockout mice lack the 2 major neurological symptoms of AHDS, namely locomotor problems and cognitive impairment. The pathological mechanism explaining the symptoms is still obscure, and no cure for this condition is known. The development of an animal model that carries MCT8-related neurological symptoms is warranted. We have employed morpholino-based gene knockdown to create zebrafish deficient for mct8. Knockdown of mct8 results in specific symptoms in the thyroid axis and brain. The mct8-morphants showed impaired locomotor behavior and brain development. More specifically, we observed maldevelopment of the cerebellum and mid-hindbrain boundary and apoptotic clusters in the zebrafish brain. The mRNA expression of zebrafish orthologs of mammalian TSH, thyroid hormone transporters, and deiodinases was altered in mct8 morphants. In particular, deiodinase type 3 gene expression was consistently up-regulated in zebrafish mct8 morphants. The thyroid hormone metabolite tetrac, but not T3, partly ameliorated the affected phenotype and locomotion disability of morphant larvae. Our results show that mct8 knockdown in zebrafish larvae results in disturbances in the thyroid axis, brain, and locomotion behavior, which is congruent with the clinical aspect of impaired locomotion and cognition in patients with AHDS. Taken together, the zebrafish is a suitable animal model for the study of the pathophysiology of AHDS.

The effects of environmental enrichment and age-related differences on inhibitory avoidance in zebrafish (Danio rerio Hamilton).

The inhibitory avoidance paradigm allows the study of mechanisms underlying learning and memory formation in zebrafish (Danio rerio Hamilton). For zebrafish, the physiology and behavior associated with this paradigm are as yet poorly understood. We therefore assessed the effects of environmental enrichment and fish age on inhibitory avoidance learning. Fish raised in an environmentally enriched tank showed decreased anxiety-like behavior and increased exploration. Enrichment greatly reduced inhibitory avoidance in 6-month (6M)- and 12-month (12 M)-old fish. Following inhibitory avoidance, telencephalic mRNA levels of proliferating cell nuclear antigen (pcna), neurogenic differentiation (neurod), cocaine- and amphetamine-regulated transcript 4 (cart4), and cannabinoid receptor 1 (cnr1) were lower in enriched-housed fish, while the ratios of mineralocorticoid receptor (nr3c2)/glucocorticoid receptor α [nr3c1(α)] and glucocorticoid receptor β [nr3c1(β)]/glucocorticoid receptor α [nr3c1(α)] were higher. This was observed for 6M-old fish only, not for 24-month (24 M) old fish. Instead, 24 M-old fish showed delayed inhibitory avoidance, no effects of enrichment, and reduced expression of neuroplasticity genes. Overall, our data show strong differences in inhibitory avoidance behavior between zebrafish of different ages and a clear reduction in avoidance behavior following housing under environmental enrichment.