During embryogenesis, organ development is dependent upon maintaining appropriate progenitor cell commitment. Synovial joints develop from a pool of progenitor cells that differentiate into various cell types constituting the mature joint. The involvement of the musculature in joint formation has long been recognized. However, the mechanism by which the musculature regulates joint formation has remained elusive. In this study, we demonstrate, utilizing various murine models devoid of limb musculature or its contraction, that the contracting musculature is fundamental in maintaining joint progenitors committed to their fate, a requirement for correct joint cavitation and morphogenesis. Furthermore, contraction-dependent activation of beta-catenin, a key modulator of joint formation, provides a molecular mechanism for this regulation. In conclusion, our findings provide the missing link between progenitor cell fate determination and embryonic movement, two processes shown to be essential for correct organogenesis.

SUMMARYAfter fertilization, the expanding carpel of fleshy fruit goes through a phase change to ripening. Although the role of ethylene signalling in mediating climacteric ripening has been established, knowledge regarding the regulation of ethylene biosynthesis and its association with fruit developmental programs is still lacking. A functional screen of tomato transcription factors showed that silencing of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS box gene results in altered fruit pigmentation. Over-expressing TAGL1 as a chimeric repressor suggested a role in controlling ripening, as transgenic tomato fruit showed reduced carotenoid and ethylene levels, suppressed chlorophyll breakdown, and down-regulation of ripening-associated genes. Moreover, fruits over-expressing TAGL1 accumulated more lycopene, and their sepals were swollen, accumulated high levels of the yellow flavonoid naringenin chalcone and contained lycopene. Transient promoter-binding assays indicated that part of the TAGL1 activity in ripening is executed through direct activation of ACS2, an ethylene biosynthesis gene that has recently been reported to be a target of the RIN MADS box factor. Examination of the TAGL1 transcript and its over-expression in the rin mutant background suggested that RIN does not regulate TAGL1 or vice versa. The results also indicated RIN-dependent and -independent processes that are regulated by TAGL1. We also noted that fruit of TAGL1 loss-of-function lines had a thin pericarp layer, indicating an additional role for TAGL1 in carpel expansion prior to ripening. The results add a new component to the current model of the regulatory network that controls fleshy fruit ripening and its association with the ethylene biosynthesis pathway.

SummaryBetalains are tyrosine-derived red-violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3-hydroxylation of tyrosine to form L-3,4-dihydroxyphenylalanine (L-DOPA).We used transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain-related cytochrome P450-type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality.L-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both L-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity.The new findings enabled us to metabolically engineer entirely red-pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.