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.

A genetic approach reveals different modes of action of prefoldins.

The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.

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.

Regulation of DELLA Proteins by Post-translational Modifications.

DELLA proteins are the negative regulators of the gibberellin (GA) signaling pathway. GAs have a pervasive effect on plant physiology, influencing processes that span the entire life cycle of the plant. All the information encoded by GAs, either environmental or developmental in origin, is canalized through DELLAs, which modulate the activity of many transcription factors and transcriptional regulators. GAs unlock the signaling pathway by triggering DELLA polyubiquitination and degradation by the 26S proteasome. Recent reports indicate, however, that there are other pathways that trigger DELLA polyubiquitination and degradation independently of GAs. Moreover, results gathered during recent years indicate that other post-translational modifications (PTMs), namely phosphorylation, SUMOylation and glycosylation, modulate DELLA function. The convergence of several PTMs in DELLA therefore highlights the strict regulation to which these proteins are subject. In this review, we summarize these discoveries and discuss DELLA PTMs from an evolutionary perspective and examine the possibilities these and other post-translational regulations offer to improve DELLA-dependent agronomic traits.

Separate elements of the TERMINAL FLOWER 1 cis-regulatory region integrate pathways to control flowering time and shoot meristem identity.

TERMINAL FLOWER 1 (TFL1) is a key regulator of Arabidopsis plant architecture that responds to developmental and environmental signals to control flowering time and the fate of shoot meristems. TFL1 expression is dynamic, being found in all shoot meristems, but not in floral meristems, with the level and distribution changing throughout development. Using a variety of experimental approaches we have analysed the TFL1 promoter to elucidate its functional structure. TFL1 expression is based on distinct cis-regulatory regions, the most important being located 3' of the coding sequence. Our results indicate that TFL1 expression in the shoot apical versus lateral inflorescence meristems is controlled through distinct cis-regulatory elements, suggesting that different signals control expression in these meristem types. Moreover, we identified a cis-regulatory region necessary for TFL1 expression in the vegetative shoot and required for a wild-type flowering time, supporting that TFL1 expression in the vegetative meristem controls flowering time. Our study provides a model for the functional organisation of TFL1 cis-regulatory regions, contributing to our understanding of how developmental pathways are integrated at the genomic level of a key regulator to control plant architecture.

Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation.

The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL is currently unknown. To better understand the regulatory relationship between LFY and AP1/CAL and to obtain deeper insights into the control of floral initiation, we monitored the activity of LFY in the absence of AP1/CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL act as part of an incoherent feed-forward loop, a network motif where two interconnected pathways or transcription factors act in opposite directions on a target gene, to control the establishment of a stable developmental program for the formation of flowers.

Clearing of Vascular Tissue in Arabidopsis thaliana for Reporter Analysis of Gene Expression.

To study the gene regulatory mechanisms modulating development is essential to visualize gene expression patterns at cellular resolution. However, this kind of analysis has been limited as a consequence of the plant tissues' opacity. In the last years, ClearSee has been increasingly used to obtain high-quality imaging of plant tissue anatomy combined with the visualization of gene expression patterns. ClearSee is established as a major tissue clearing technique due to its simplicity and versatility.In this chapter, we outline an easy-to-follow ClearSee protocol to analyze gene expression of reporters using either ß-glucuronidase (GUS) or fluorescent protein (FP) tags, compatible with different dyes to stain cell walls. We detail materials, equipment, solutions, and procedures to easily implement ClearSee for the study of vascular development in Arabidopsis thaliana, but the protocol can be easily adapted to a variety of plant tissues in a wide range of plant species.

Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha.

Plant survival depends on the optimal use of resources under variable environmental conditions. Among the mechanisms that mediate the balance between growth, differentiation, and stress responses, the regulation of transcriptional activity by DELLA proteins stands out. In angiosperms, DELLA accumulation promotes defense against biotic and abiotic stress and represses cell division and expansion, while the loss of DELLA function is associated with increased plant size and sensitivity toward stress.1 Given that DELLA protein stability is dependent on gibberellin (GA) levels2 and GA metabolism is influenced by the environment,3 this pathway is proposed to relay environmental information to the transcriptional programs that regulate growth and stress responses in angiosperms.4,5 However, DELLA genes are also found in bryophytes, whereas canonical GA receptors have been identified only in vascular plants.6-10 Thus, it is not clear whether these regulatory functions of DELLA predated or emerged with typical GA signaling. Here, we show that, as in vascular plants, the only DELLA in the liverwort Marchantia polymorpha also participates in the regulation of growth and key developmental processes and promotes oxidative stress tolerance. Moreover, part of these effects is likely caused by the conserved physical interaction with the MpPIF transcription factor. Therefore, we suggest that the role in the coordination of growth and stress responses was already encoded in the DELLA protein of the common ancestor of land plants, and the importance of this function is underscored by its conservation over the past 450 million years.

Cauliflower fractal forms arise from perturbations of floral gene networks.

Throughout development, plant meristems regularly produce organs in defined spiral, opposite, or whorl patterns. Cauliflowers present an unusual organ arrangement with a multitude of spirals nested over a wide range of scales. How such a fractal, self-similar organization emerges from developmental mechanisms has remained elusive. Combining experimental analyses in an Arabidopsis thaliana cauliflower-like mutant with modeling, we found that curd self-similarity arises because the meristems fail to form flowers but keep the "memory" of their transient passage in a floral state. Additional mutations affecting meristem growth can induce the production of conical structures reminiscent of the conspicuous fractal Romanesco shape. This study reveals how fractal-like forms may emerge from the combination of key, defined perturbations of floral developmental programs and growth dynamics.

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole.

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for "modified traffic to the vacuole") mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative delta-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.

The pillars of land plants: new insights into stem development.

In spite of its central importance in evolution, plant architecture and crop improvement, stem development remains poorly understood relative to other plant organs. Here, we summarise current knowledge of stem ontogenesis and its regulation, including insights from new image analysis and biophysical approaches. The stem initiates in the rib zone (RZ) of the shoot apical meristem, under transcriptional control by DELLA and BLH proteins. Links have emerged between these regulators and cell proliferation, patterning and oriented growth in the RZ. During subsequent internode elongation, cell wall properties and mechanics have been analysed in detail, revealing pectin modification as a prominent control point. Recent work has also highlighted signalling to coordinate growth of stem tissues with different mechanical properties.

DELLA genes restrict inflorescence meristem function independently of plant height.

DELLA proteins associate with transcription factors to control plant growth in response to gibberellin 1 . Semi-dwarf DELLA mutants with improved harvest index and decreased lodging greatly improved global food security during the 'green revolution' in the 1960-1970s 2 . However, DELLA mutants are pleiotropic and the developmental basis for their effects on plant architecture remains poorly understood. Here, we show that DELLA proteins have genetically separable roles in controlling stem growth and the size of the inflorescence meristem, where flowers initiate. Quantitative three-dimensional image analysis, combined with a genome-wide screen for DELLA-bound loci in the inflorescence tip, revealed that DELLAs limit meristem size in Arabidopsis by directly upregulating the cell-cycle inhibitor KRP2 in the underlying rib meristem, without affecting the canonical WUSCHEL-CLAVATA meristem size regulators 3 . Mutation of KRP2 in a DELLA semi-dwarf background restored meristem size, but not stem growth, and accelerated flower production. In barley, secondary mutations in the DELLA gain-of-function mutant Sln1d 4 also uncoupled meristem and inflorescence size from plant height. Our work reveals an unexpected and conserved role for DELLA genes in controlling shoot meristem function and suggests how dissection of pleiotropic DELLA functions could unlock further yield gains in semi-dwarf mutants.

Regulatory interplay between LEAFY, APETALA1/CAULIFLOWER and TERMINAL FLOWER1: New insights into an old relationship.

The gene regulatory network comprised of LEAFY (LFY), APETALA1 (AP1), the AP1 paralog CAULIFLOWER (CAL), and TERMINAL FLOWER1 (TFL1) is a major determinant of the flowering process in Arabidopsis thaliana. TFL1 activity in the shoot apical meristem provides inflorescence identity while the transcription factors LFY and AP1/CAL confer floral identity to emerging floral primordia. It has been thought that LFY and AP1/CAL control the onset of flowering in part by repressing TFL1 expression in flowers. However, in the June issue of Plant Physiology, we reported that LFY and AP1 act antagonistically in the regulation of several key flowering regulators, including TFL1. Specifically, TFL1 transcription was suppressed by AP1 but promoted by LFY. Here, we present additional evidence for the role of LFY as an activator of TFL1 and propose that this regulatory activity is pivotal for the indeterminate growth of the SAM during the reproductive phase of development.

Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis.

The origin of the stem is a major but poorly understood aspect of plant development, partly because the stem initiates in a relatively inaccessible region of the shoot apical meristem called the rib zone (RZ). We developed quantitative 3D image analysis and clonal analysis tools, which revealed that the Arabidopsis homeodomain protein REPLUMLESS (RPL) establishes distinct patterns of oriented cell division and growth in the central and peripheral regions of the RZ. A genome-wide screen for target genes connected RPL directly to many of the key shoot development pathways, including the development of organ boundaries; accordingly, mutation of the organ boundary gene LIGHT-SENSITIVE HYPOCOTYL 4 restored RZ function and stem growth in the rpl mutant. Our work opens the way to study a developmental process of importance to crop improvement and highlights how apparently simple changes in 3D organ growth can reflect more complex internal changes in oriented cell activities.

Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis.

How cells regulate their dimensions is a long-standing question. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle, although there is evidence of cell-autonomous control in cell cultures. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures.

Orchestration of floral initiation by APETALA1.

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways.

Floral initiation and inflorescence architecture: a comparative view.

BACKGROUND: A huge variety of plant forms can be found in nature. This is particularly noticeable for inflorescences, the region of the plant that contains the flowers. The architecture of the inflorescence depends on its branching pattern and on the relative position where flowers are formed. In model species such as Arabidopsis thaliana or Antirrhinum majus the key genes that regulate the initiation of flowers have been studied in detail and much is known about how they work. Studies being carried out in other species of higher plants indicate that the homologues of these genes are also key regulators of the development of their reproductive structures. Further, changes in these gene expression patterns and/or function play a crucial role in the generation of different plant architectures. SCOPE: In this review we aim to present a summarized view on what is known about floral initiation genes in different plants, particularly dicotyledonous species, and aim to emphasize their contribution to plant architecture.