Harnessing clonal gametes in hybrid crops to engineer polyploid genomes.

Heterosis boosts crop yield; however, harnessing additional progressive heterosis in polyploids is challenging for breeders. We bioengineered a 'mitosis instead of meiosis' (MiMe) system that generates unreduced, clonal gametes in three hybrid tomato genotypes and used it to establish polyploid genome design. Through the hybridization of MiMe hybrids, we generated '4-haplotype' plants that encompassed the complete genetics of their four inbred grandparents, providing a blueprint for exploiting polyploidy in crops.

AGAMOUS mediates timing of guard cell formation during gynoecium development.

In Arabidopsis thaliana, stomata are composed of two guard cells that control the aperture of a central pore to facilitate gas exchange between the plant and its environment, which is particularly important during photosynthesis. Although leaves are the primary photosynthetic organs of flowering plants, floral organs are also photosynthetically active. In the Brassicaceae, evidence suggests that silique photosynthesis is important for optimal seed oil content. A group of transcription factors containing MADS DNA binding domains is necessary and sufficient to confer floral organ identity. Elegant models, such as the ABCE model of flower development and the floral quartet model, have been instrumental in describing the molecular mechanisms by which these floral organ identity proteins govern flower development. However, we lack a complete understanding of how the floral organ identity genes interact with the underlying leaf development program. Here, we show that the MADS domain transcription factor AGAMOUS (AG) represses stomatal development on the gynoecial valves, so that maturation of stomatal complexes coincides with fertilization. We present evidence that this regulation by AG is mediated by direct transcriptional repression of a master regulator of the stomatal lineage, MUTE, and show data that suggests this interaction is conserved among several members of the Brassicaceae. This work extends our understanding of the mechanisms underlying floral organ formation and provides a framework to decipher the mechanisms that control floral organ photosynthesis.

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

PIF4 enhances DNA binding of CDF2 to co-regulate target gene expression and promote Arabidopsis hypocotyl cell elongation.

How specificity is conferred within gene regulatory networks is an important problem in biology. The basic helix-loop-helix PHYTOCHROME-INTERACTING FACTORs (PIFs) and single zinc-finger CYCLING DOF FACTORs (CDFs) mediate growth responses of Arabidopsis to light and temperature. We show that these two classes of transcription factor (TF) act cooperatively. CDF2 and PIF4 are temporally and spatially co-expressed, they interact to form a protein complex and act in the same genetic pathway to promote hypocotyl cell elongation. Furthermore, PIF4 substantially strengthens genome-wide occupancy of CDF2 at a subset of its target genes. One of these, YUCCA8, encodes an auxin biosynthesis enzyme whose transcription is increased by PIF4 and CDF2 to contribute to hypocotyl elongation. The binding sites of PIF4 and CDF2 in YUCCA8 are closely spaced, and in vitro PIF4 enhances binding of CDF2. We propose that this occurs by direct protein interaction and because PIF4 binding alters DNA conformation. Thus, we define mechanisms by which PIF and CDF TFs cooperate to achieve regulatory specificity and promote cell elongation in response to light.

MicroRNA172 controls inflorescence meristem size through regulation of APETALA2 in Arabidopsis.

The APETALA2 (AP2) transcription factor regulates flower development, floral transition and shoot apical meristem (SAM) maintenance in Arabidopsis. AP2 is also regulated at the post-transcriptional level by microRNA172 (miR172), but the contribution of this to SAM maintenance is poorly understood. We generated transgenic plants carrying a form of AP2 that is resistant to miR172 (rAP2) or carrying a wild-type AP2 susceptible to miR172. Phenotypic and genetic analyses were performed on these lines and mir172 mutants to study the role of AP2 regulation by miR172 on meristem size and the rate of flower production. We found that rAP2 enlarges the inflorescence meristem by increasing cell size and cell number. Misexpression of rAP2 from heterologous promoters showed that AP2 acts in the central zone (CZ) and organizing center (OC) to increase SAM size. Furthermore, we found that AP2 is negatively regulated by AUXIN RESPONSE FACTOR 3 (ARF3). However, genetic analyses indicated that ARF3 also influences SAM size and flower production rate independently of AP2. The study identifies miR172/AP2 as a regulatory module controlling inflorescence meristem size and suggests that transcriptional regulation of AP2 by ARF3 fine-tunes SAM size determination.

INTERMEDIUM-M encodes an HvAP2L-H5 ortholog and is required for inflorescence indeterminacy and spikelet determinacy in barley.

Inflorescence architecture dictates the number of flowers and, ultimately, seeds. The architectural discrepancies between two related cereals, barley and wheat, are controlled by differences in determinacy of inflorescence and spikelet meristems. Here, we characterize two allelic series of mutations named intermedium-m (int-m) and double seed1 (dub1) that convert barley indeterminate inflorescences into wheat-like determinate inflorescences bearing a multifloreted terminal spikelet and spikelets with additional florets. INT-M/DUB1 encodes an APETALA2-like transcription factor (HvAP2L-H5) that suppresses ectopic and precocious spikelet initiation signals and maintains meristem activity. HvAP2L-H5 inhibits the identity shift of an inflorescence meristem (IM) to a terminal spikelet meristem (TSM) in barley. Null mutations in AP2L-5 lead to fewer spikelets per inflorescence but extra florets per spikelet. In wheat, prolonged and elevated AP2L-A5 activity in rAP2L-A5 mutants delays but does not suppress the IM-TSM transition. We hypothesize that the regulation of AP2L-5 orthologs and downstream genes contributes to the different inflorescence determinacy in barley and wheat. We show that AP2L-5 proteins are evolutionarily conserved in grasses, promote IM activity, and restrict floret number per spikelet. This study provides insights into the regulation of spikelet and floret number, and hence grain yield in barley and wheat.

The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner.

SWI/SNF ATP-dependent chromatin remodeling complexes (CRCs) play important roles in the regulation of transcription, cell cycle, DNA replication, repair, and hormone signaling in eukaryotes. The core of SWI/SNF CRCs composed of a SWI2/SNF2 type ATPase, a SNF5 and two of SWI3 subunits is sufficient for execution of nucleosome remodeling in vitro. The Arabidopsis genome encodes four SWI2/SNF2 ATPases, four SWI3, a single SNF5 and two SWP73 subunits. Genes of the core SWI/SNF components have critical but not fully overlapping roles during plant growth, embryogenesis, and sporophyte development. Here we show that the Arabidopsis swi3c mutant exhibits a phenotypic reversion when grown at lower temperature resulting in partial restoration of its embryo, root development and fertility defects. Our data indicates that the swi3c mutation alters the expression of several genes engaged in low temperature responses. The location of SWI3C-containing SWI/SNF CRCs on the ICE1, MYB15 and CBF1 target genes depends on the temperature conditions, and the swi3c mutation thus also influences the transcription of several cold-responsive (COR) genes. These findings, together with genetic analysis of swi3c/ice1 double mutant and enhanced freezing tolerance of swi3c plants illustrate that SWI/SNF CRCs contribute to fine-tuning of plant growth responses to different temperature regimes.

Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions.

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development.

Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control.

p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

DELLA-interacting SWI3C core subunit of switch/sucrose nonfermenting chromatin remodeling complex modulates gibberellin responses and hormonal cross talk in Arabidopsis.

Switch (SWI)/Sucrose Nonfermenting (SNF)-type chromatin-remodeling complexes (CRCs) are involved in regulation of transcription, DNA replication and repair, and cell cycle. Mutations of conserved subunits of plant CRCs severely impair growth and development; however, the underlying causes of these phenotypes are largely unknown. Here, we show that inactivation of SWI3C, the core component of Arabidopsis (Arabidopsis thaliana) SWI/SNF CRCs, interferes with normal functioning of several plant hormone pathways and alters transcriptional regulation of key genes of gibberellin (GA) biosynthesis. The resulting reduction of GA4 causes severe inhibition of hypocotyl and root elongation, which can be rescued by exogenous GA treatment. In addition, the swi3c mutation inhibits DELLA-dependent transcriptional activation of GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptor genes. Down-regulation of GID1a in parallel with the DELLA repressor gene REPRESSOR OF GA1-3 1 in swi3c indicates that lack of SWI3C also leads to defects in GA signaling. Together with the recent demonstration of function of SWI/SNF ATPase BRAHMA in the GA pathway, these results reveal a critical role of SWI/SNF CRC in the regulation of GA biosynthesis and signaling. Moreover, we demonstrate that SWI3C is capable of in vitro binding to, and shows in vivo bimolecular fluorescence complementation interaction in cell nuclei with, the DELLA proteins RGA-LIKE2 and RGA-LIKE3, which affect transcriptional activation of GID1 and GA3ox (GIBBERELLIN 3-OXIDASE) genes controlling GA perception and biosynthesis, respectively. Furthermore, we show that SWI3C also interacts with the O-GlcNAc (O-linked N-acetylglucosamine) transferase SPINDLY required for proper functioning of DELLAs and acts hypostatically to (SPINDLY) in the GA response pathway. These findings suggest that DELLA-mediated effects in GA signaling as well as their role as a hub in hormonal cross talk may be, at least in part, dependent on their direct physical interaction with complexes responsible for modulation of chromatin structure.

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis.

Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata.