DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants.

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

The prefoldin-like protein AtURI exhibits characteristics of intrinsically disordered proteins.

The prefoldin-like protein UNCONVENTIONAL PREFOLDIN RPB5 INTERACTOR (URI) participates in diverse cellular functions, including protein homeostasis, transcription, translation, and signal transduction. Thus, URI is a highly versatile protein, although the molecular basis of this versatility remains unknown. In this work, we show that Arabidopsis thaliana (Arabidopsis) URI (AtURI) possesses a large intrinsically disordered region (IDR) spanning most of the C-terminal part of the protein, a feature conserved in yeast and human orthologs. Our findings reveal two key characteristics of disordered proteins in AtURI: promiscuity in interacting with partners and protein instability. We propose that these two features contribute to providing AtURI with functional versatility.

Identification of MeC3HDZ1/MeCNA as a potential regulator of cassava storage root development.

The storage root (SR) of cassava is the main staple food in sub-Saharan Africa, where it feeds over 500 million people. However, little is known about the genetic and molecular regulation underlying its development. Unraveling such regulation would pave the way for biotechnology approaches aimed at enhancing cassava productivity. Anatomical studies indicate that SR development relies on the massive accumulation of xylem parenchyma, a cell-type derived from the vascular cambium. The C3HDZ family of transcription factors regulate cambial cells proliferation and xylem differentiation in Arabidopsis and other species. We thus aimed at identifying C3HDZ proteins in cassava and determining whether any of them shows preferential activity in the SR cambium and/or xylem. Using phylogeny and synteny studies, we identified eight C3HDZ proteins in cassava, namely MeCH3DZ1-8. We observed that MeC3HDZ1 is the MeC3HDZ gene displaying the highest expression in SR and that, within that organ, the gene also shows high expression in cambium and xylem. In-silico analyses revealed the existence of a number of potential C3HDZ targets displaying significant preferential expression in the SR. Subsequent Y1H analyses proved that MeC3HDZ1 can bind canonical C3HDZ binding sites, present in the promoters of these targets. Transactivation assays demonstrated that MeC3HDZ1 can regulate the expression of genes downstream of promoters harboring such binding sites, thereby demonstrating that MeC3HDZ1 has C3HDZ transcription factor activity. We conclude that MeC3HDZ1 may be a key factor for the regulation of storage root development in cassava, holding thus great promise for future biotechnology applications.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Quantification of Tracheary Elements Types in Mature Hypocotyl of Arabidopsis thaliana.

Secondary growth is a highly relevant process for dicot and gymnosperm species development. The process relies on vascular tissue proliferation and culminates with the thickening of stems, roots, and hypocotyls. The formation of tracheary elements is a critical step during this process. Among such tracheary elements, four different cell types are distinguished depending on their secondary cell wall pattern, which is exclusive for each tracheary cell type. Here we describe a method to isolate, dye, and recognize each of these tracheary cell types. The method is optimized to be performed in the Arabidopsis thaliana hypocotyl. This is because, in this species, the hypocotyl is the organ undergoing the largest proportion of secondary growth. Results allow for determining the relative amounts of each of the tracheary cell types.

DELLA functions evolved by rewiring of associated transcriptional networks.

DELLA proteins are land-plant specific transcriptional regulators that transduce environmental information to multiple processes throughout a plant's life1-3. The molecular basis for this critical function in angiosperms has been linked to the regulation of DELLA stability by gibberellins and to the capacity of DELLA proteins to interact with hundreds of transcription factors4,5. Although bryophyte orthologues can partially fulfil functions attributed to angiosperm DELLA6,7, it is not clear whether the capacity to establish interaction networks is an ancestral property of DELLA proteins or is associated with their role in gibberellin signalling8-10. Here we show that representative DELLAs from the main plant lineages display a conserved ability to interact with multiple transcription factors. We propose that promiscuity was encoded in the ancestral DELLA protein, and that this property has been largely maintained, whereas the lineage-dependent diversification of DELLA-dependent functions mostly reflects the functional evolution of their interacting partners.

Prefoldins contribute to maintaining the levels of the spliceosome LSM2-8 complex through Hsp90 in Arabidopsis.

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2-8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2-8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2-8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.

SOBIR1/EVR prevents precocious initiation of fiber differentiation during wood development through a mechanism involving BP and ERECTA.

In plants, secondary growth results in radial expansion of stems and roots, generating large amounts of biomass in the form of wood. Using genome-wide association studies (GWAS)-guided reverse genetics in Arabidopsis thaliana, we discovered SOBIR1/EVR, previously known to control plant immunoresponses and abscission, as a regulator of secondary growth. We present anatomical, genetic, and molecular evidence indicating that SOBIR1/EVR prevents the precocious differentiation of xylem fiber, a key cell type for wood development. SOBIR1/EVR acts through a mechanism that involves BREVIPEDICELLUS (BP) and ERECTA (ER), 2 proteins previously known to regulate xylem fiber development. We demonstrate that BP binds SOBIR1/EVR promoter and that SOBIR1/EVR expression is enhanced in bp mutants, suggesting a direct, negative regulation of BP over SOBIR1/EVR expression. We show that SOBIR1/EVR physically interacts with ER and that defects caused by the sobir1/evr mutation are aggravated by mutating ER, indicating that SOBIR1/EVR and ERECTA act together in the control of the precocious formation of xylem fiber development.

Regulation of xylem fiber differentiation by gibberellins through DELLA-KNAT1 interaction.

The thickening of plant organs is supported by secondary growth, a process by which new vascular tissues (xylem and phloem) are produced. Xylem is composed of several cell types, including xylary fibers, parenchyma and vessel elements. In Arabidopsis, it has been shown that fibers are promoted by the class-I KNOX gene KNAT1 and the plant hormones gibberellins, and are repressed by a small set of receptor-like kinases; however, we lack a mechanistic framework to integrate their relative contributions. Here, we show that DELLAs, negative elements of the gibberellin signaling pathway, physically interact with KNAT1 and impair its binding to KNAT1-binding sites. Our analysis also indicates that at least 37% of the transcriptome mobilized by KNAT1 is potentially dependent on this interaction, and includes genes involved in secondary cell wall modifications and phenylpropanoid biosynthesis. Moreover, the promotion by constitutive overexpression of KNAT1 of fiber formation and the expression of genes required for fiber differentiation were still reverted by DELLA accumulation, in agreement with post-translational regulation of KNAT1 by DELLA proteins. These results suggest that gibberellins enhance fiber development by promoting KNAT1 activity.

A bHLH-Based Feedback Loop Restricts Vascular Cell Proliferation in Plants.

Control of tissue dimensions in multicellular organisms requires the precise quantitative regulation of mitotic activity. In plants, where cells are immobile, tissue size is achieved through control of both cell division orientation and mitotic rate. The bHLH transcription factor heterodimer formed by target of monopteros5 (TMO5) and lonesome highway (LHW) is a central regulator of vascular width-increasing divisions. An important unanswered question is how its activity is limited to specify vascular tissue dimensions. Here we identify a regulatory network that restricts TMO5/LHW activity. We show that thermospermine synthase ACAULIS5 antagonizes TMO5/LHW activity by promoting the accumulation of SAC51-LIKE (SACL) bHLH transcription factors. SACL proteins heterodimerize with LHW-therefore likely competing with TMO5/LHW interactions-prevent activation of TMO5/LHW target genes, and suppress the over-proliferation caused by excess TMO5/LHW activity. These findings connect two thus-far disparate pathways and provide a mechanistic understanding of the quantitative control of vascular tissue growth.

Role of the gibberellin receptors GID1 during fruit-set in Arabidopsis.

Gibberellins (GAs) play a critical role in fruit-set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit-set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue-specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C-RGL1 and GID1B-RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1-DELLA in the different GA-dependent processes that occur upon fruit-set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.

Characterization of constricted fruit (ctf) mutant uncovers a role for AtMYB117/LOF1 in ovule and fruit development in Arabidopsis thaliana.

Pistil and fruit morphogenesis is the result of a complex gene network that is not yet fully understood. A search for novel genes is needed to make a more comprehensive model of pistil and fruit development. Screening for mutants with alterations in fruit morphology generated by an activation tagging strategy resulted in the isolation of the ctf (constricted fruit) mutant. It is characterized by a) small and wrinkled fruits, with an enlarged replum, an amorphous structure of the septum and an irregular distribution of ovules and seeds; b) ectopic carpelloid structures in sepals bearing ovule-like structures and c) dwarf plants with curled rosette leaves. The overexpressed gene in ctf was AtMYB117, also named LOF1 (LATERAL ORGAN FUSION1). AtMYB117/LOF1 transcripts were localized in boundary regions of the vegetative shoot apical meristem and leaf primordia and in a group of cells in the adaxial base of petioles and bracts. Transcripts were also detected in the boundaries between each of the four floral whorls and during pistil development in the inner of the medial ridges, the placenta, the base of the ovule primordia, the epidermis of the developing septum and the outer cell layers of the ovule funiculi. Analysis of changes of expression of pistil-related genes in the ctf mutant showed an enhancement of SHATTERPROOF1 (SHP1) and SHP2 expression. All these results suggest that AtMYB117/LOF1 is recruited by a variety of developmental programs for the establishment of boundary regions, including the development of floral organs and the initiation of ovule outgrowth.

Ethylene is involved in pistil fate by modulating the onset of ovule senescence and the GA-mediated fruit set in Arabidopsis.

BACKGROUND: Ovule lifespan is an important factor in determining the ability to set fruits and produce seeds. Once ovule senescence is established, fruit set capacity in response to gibberellins (GAs) is lost. We aimed to elucidate whether ethylene plays a role in controlling ovule senescence and the fruit set response in Arabidopsis. RESULTS: Ethylene response inhibitors, silver thiosulphate (STS) and 1-methylcyclopropene (1-MCP), were able to delay the loss of pistil response to GA(3). In addition, ethylene insensitive mutants ein2-5 and ein3-1 showed delayed loss of pistil response, as in plants treated with STS and 1-MCP, while constitutive mutant ctr1-1 displayed premature loss of response. The analysis of the expression of ethylene biosynthesis genes suggests that ethylene is synthesised in ovules at the onset of ovule senescence, while a transcriptional meta-analysis also supports an activated ethylene-dependent senescence upon the establishment of ovule senescence. Finally, a SAG12:GUS reporter line proved useful to monitor ovule senescence and to directly demonstrate that ethylene specifically modulates ovule senescence. CONCLUSIONS: We have shown that ethylene is involved in both the control of the ovule lifespan and the determination of the pistil/fruit fate. Our data support a role of the ovule in modulating the GA response during fruit set in Arabidopsis. A possible mechanism that links the ethylene modulation of the ovule senescence and the GA3-induced fruit set response is discussed.

A fertilization-independent developmental program triggers partial fruit development and senescence processes in pistils of Arabidopsis.

The pistil is the specialized plant organ that enables appropriate pollination and ovule fertilization, after which it undergoes growth and differentiation to become a fruit. However, in most species, if ovules are not fertilized around anthesis the pistil irreversibly loses its growth capacity. We used physiological, molecular, and transcriptomic tools to characterize the post-anthesis development of the unfertilized Arabidopsis (Arabidopsis thaliana) pistil. Surprisingly, developmental processes that have been previously described in developing Arabidopsis fruits, such as the collapse of the adaxial epidermis, differentiation of a sclerenchyma layer in the adaxial subepidermis and the dehiscence zone, and valve dehiscence, were also observed in the unfertilized pistil. We determined that senescence is first established in the transmitting tract, stigma, and ovules immediately after anthesis, and that the timing of senescence in the stigma and ovules correlates with the loss of fruit-set responsiveness of the pistil to pollen and the hormone gibberellin (GA), respectively. Moreover, we showed that mutants with altered ovule development have impaired fruit-set response to the GA gibberellic acid, which further indicates that the presence of viable ovules is required for fruit-set responsiveness to GAs in the unfertilized pistil. Our data suggest that a fertilization-independent developmental program controls many of the processes during post-anthesis development, both in unfertilized pistils and seeded fruits, and point to a key role of the ovule in the capacity of pistils to undergo fruit set in response to GA.

Fertilization-dependent auxin response in ovules triggers fruit development through the modulation of gibberellin metabolism in Arabidopsis.

Fruit development is usually triggered by ovule fertilization, and it requires coordination between seed development and the growth and differentiation of the ovary to host the seeds. Hormones are known to synchronize these two processes, but the role of each hormone, and the mechanism by which they interact, are still unknown. Here we show that auxin and gibberellins (GAs) act in a hierarchical scheme. The synthetic reporter construct DR5:GFP showed that fertilization triggered an increase in auxin response in the ovules, which could be mimicked by blocking polar auxin transport. As the application of GAs did not affect auxin response, the most likely sequence of events after fertilization involves auxin-mediated activation of GA synthesis. We have confirmed this, and have shown that GA biosynthesis upon fertilization is localized specifically in the fertilized ovules. Furthermore, auxin treatment caused changes in the expression of GA biosynthetic genes similar to those triggered by fertilization, and also restricted to the ovules. Finally, GA signaling was activated in ovules and valves, as shown by the rapid downregulation of the fusion protein RGA-GFP after pollination and auxin treatment. Taken together, this evidence suggests a model in which fertilization would trigger an auxin-mediated promotion of GA synthesis specifically in the ovule. The GAs synthesized in the ovules would be then transported to the valves to promote GA signaling and thus coordinate growth of the silique.

Expression of PsGRP1, a novel glycine rich protein gene of Pisum sativum, is induced in developing fruit and seed and by ABA in pistil and root.

A novel glycine-rich protein gene, PsGRP1, has been identified in Pisum sativum L. Accumulation of PsGRP1 transcripts was observed in reproductive organs and vegetative tissues. They were localized in endocarp sclerenchyma during fruit development in cells that will lignify. PsGRP1 expression was also detected in senescent pistils and developing seeds and induced by ABA treatment in presenescent pistils. A raise in the expression was also observed in roots after treatment with ABA or mannitol but not under cold stress. A mannitol treatment induced a rise in ABA levels and fluridone treatment counteracted the mannitol induction of PsGRP1 expression. The results suggest a possible role for PsGRP1 in differentiation of the endocarp sclerenchyma and during seed development, pistil senescence and osmotic stress under ABA control.

A serine carboxypeptidase gene (PsCP), expressed in early steps of reproductive and vegetative development in Pisum sativum, is induced by gibberellins.

A cDNA clone encoding a serine carboxypeptidase (PsCP), isolated from young fruits of Pisum sativum L., was used to study the temporal and spatial expression and hormonal regulation of serine carboxypeptidase during reproductive and vegetative development. In unpollinated pea ovaries PsCP transcript levels decreased during senescence. However, during early fruit development, PsCP transcript were accumulated in both pericarp and seeds, preferentially in the nucellus, with a polar distribution at the chalazal region of the embryo sac, suggesting a role in seed development. PsCP transcript levels increased also when fruit set was induced in unpollinated ovaries by gibberellins, although the distribution was uniform. PsCP expression was also induced by auxins but not cytokinins, indicating a selective hormonal regulation of PsCP transcription. Localization of PsCP transcript after pollination parallel reported changes in gibberellin distribution, suggesting that PsCP transcription in developing fruits and seeds is induced by gibberellins. PsCP is also expressed in developing seedlings but not in cotyledons, suggesting that it is not involved in the mobilization of storage materials. PsCP transcripts were suppressed by treatment of seedlings with paclobutrazol and restored by gibberellic acid (GA3) treatment. In addition, PsCP transcript levels decreased in etiolated pea seedlings when they were exposed to continuous light but not when exposed to light in the presence of GA3. These results indicate that PsCP transcript accumulation is induced by gibberellins in developing seedlings. This is the first report of a serine carboxypeptidase-like gene induced by gibberellins in reproductive and vegetative developing tissues in dicotyledoneous plants.