Involvement of the tomato BBX16 and BBX17 microProteins in reproductive development.

BBXs are B-Box zinc finger proteins that can act as transcription factors and regulators of protein complexes. Several BBX proteins play important roles in plant development. Two Arabidopsis thaliana microProteins belonging to the BBX family, named miP1a and miP1b, homotypically interact with and modulate the activity of other BBX proteins, including CONSTANS, which transcriptionally activates the florigen, FLOWERING LOCUS T. Arabidopsis plants overexpressing miP1a and miP1b showed delayed flowering. In tomato, the closest homologs of miP1a and miP1b are the microProteins SlBBX16 and SlBBX17. This study was aimed at investigating whether the constitutive expression of SlBBX16/17 in Arabidopsis and tomato impacted reproductive development. The heterologous expression of the two tomato microProteins in Arabidopsis caused a delay in the flowering transition; however, the effect was weaker than that observed when the native miP1a/b were overexpressed. In tomato, overexpression of SlBBX17 prolonged the flowering period; this effect was accompanied by downregulation of the flowering inhibitors Self Pruning (SP) and SP5G. SlBBX16 and SlBBX17 can hetero-oligomerize with TCMP-2, a cystine-knot peptide involved in flowering pattern regulation and early fruit development in tomato. The increased expression of both microProteins also caused alterations in tomato fruit development: we observed in the case of SlBBX17 a decrease in the number and size of ripe fruits as compared to WT plants, while for SlBBX16, a delay in fruit production up to the breaker stage. These effects were associated with changes in the expression of GA-responsive genes.

ABI5 binding proteins: key players in coordinating plant growth and development.

During the course of terrestrial evolution, plants have developed complex networks that involve the coordination of phytohormone signalling pathways in order to adapt to an ever-changing environment. Transcription factors coordinate these responses by engaging in different protein complexes and exerting both positive and negative effects. ABA INSENSITIVE 5 (ABI5) binding proteins (AFPs), which are closely related to NOVEL INTERACTOR OF JAZ (NINJA)-like proteins, are known for their fundamental role in plants' morphological and physiological growth. Recent studies have shown that AFPs regulate several hormone-signalling pathways, including abscisic acid (ABA) and gibberellic acid (GA). Here, we review the genetic control of AFPs and their crosstalk with plant hormone signalling, and discuss the contributions of AFPs to plants' growth and development.

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

FIONA1-mediated methylation of the 3'UTR of FLC affects FLC transcript levels and flowering in Arabidopsis.

Adenosine bases of RNA can be transiently modified by the deposition of a methyl-group to form N6-methyladenosine (m6A). This adenosine-methylation is an ancient process and the enzymes involved are evolutionary highly conserved. A genetic screen designed to identify suppressors of late flowering transgenic Arabidopsis plants overexpressing the miP1a microProtein yielded a new allele of the FIONA1 (FIO1) m6A-methyltransferase. To characterize the early flowering phenotype of fio1 mutant plants we employed an integrative approach of mRNA-seq, Nanopore direct RNA-sequencing and meRIP-seq to identify differentially expressed transcripts as well as differentially methylated RNAs. We provide evidence that FIO1 is the elusive methyltransferase responsible for the 3'-end methylation of the FLOWERING LOCUS C (FLC) transcript. Furthermore, our genetic and biochemical data suggest that 3'-methylation stabilizes FLC mRNAs and non-methylated FLC is a target for rapid degradation.

Context-specific functions of transcription factors controlling plant development: From leaves to flowers.

Plant development is regulated by transcription factors that often act in more than one process and stage of development. Yet the molecular mechanisms that govern the functional diversity and specificity of these proteins remains far from understood. Flower development provides an ideal context to study these mechanisms since the development of distinct floral organs depends on similar but distinct combinations of transcriptional regulators. Recent work also highlights the importance of leaf polarity regulators as additional key factors in flower initiation, floral organ morphogenesis, and possibly floral organ positioning. A detailed understanding of how these factors work in combination will enable us to address outstanding questions in flower development including how distinct shapes and positions of floral organs are generated. Experimental approaches and computer-based modeling will be required to characterize gene-regulatory networks at the level of single cells.

The genetic interaction of REVOLUTA and WRKY53 links plant development, senescence, and immune responses.

In annual plants, tight coordination of successive developmental events is of primary importance to optimize performance under fluctuating environmental conditions. The recent finding of the genetic interaction of WRKY53, a key senescence-related gene with REVOLUTA, a master regulator of early leaf patterning, raises the question of how early and late developmental events are connected. Here, we investigated the developmental and metabolic consequences of an alteration of the REVOLUTA and WRKY53 gene expression, from seedling to fruiting. Our results show that REVOLUTA critically controls late developmental phases and reproduction while inversely WRKY53 determines vegetative growth at early developmental stages. We further show that these regulators of distinct developmental phases frequently, but not continuously, interact throughout ontogeny and demonstrated that their genetic interaction is mediated by the salicylic acid (SA). Moreover, we showed that REVOLUTA and WRKY53 are keys regulatory nodes of development and plant immunity thought their role in SA metabolic pathways, which also highlights the role of REV in pathogen defence. Together, our findings demonstrate how late and early developmental events are tightly intertwined by molecular hubs. These hubs interact with each other throughout ontogeny, and participate in the interplay between plant development and immunity.

Stop CRYing! Inhibition of cryptochrome function by small proteins.

Plants can detect the presence of light using specialised photoreceptor proteins. These photoreceptors measure the intensity of light, but they can also respond to different spectra of light and thus 'see' different colours. Cryptochromes, which are also present in animals, are flavin-based photoreceptors that enable plants to detect blue and ultraviolet-A (UV-A) light. In Arabidopsis, there are two cryptochromes, CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2 (CRY2) with known sensory roles. They function in various processes such as blue-light mediated inhibition of hypocotyl elongation, photoperiodic promotion of floral initiation, cotyledon expansion, anthocyanin production, and magnetoreception, to name a few. In the dark, the cryptochromes are in an inactive monomeric state and undergo photochemical and conformational change in response to illumination. This results in flavin reduction, oligomerisation, and the formation of the 'cryptochrome complexome'. Mechanisms of cryptochrome activation and signalling have been extensively studied and found to be conserved across phylogenetic lines. In this review, we will therefore focus on a far lesser-known mechanism of regulation that is unique to plant cryptochromes. This involves inhibition of cryptochrome activity by small proteins that prevent its dimerisation in response to light. The resulting inhibition of function cause profound alterations in economically important traits such as plant growth, flowering, and fruit production. This review will describe the known mechanisms of cryptochrome activation and signalling in the context of their modulation by these endogenous and artificial small inhibitor proteins. Promising new applications for biotechnological and agricultural applications will be discussed.

Controlling flowering of Medicago sativa (alfalfa) by inducing dominant mutations.

Breeding plants with polyploid genomes is challenging because functional redundancy hampers the identification of loss-of-function mutants. Medicago sativa is tetraploid and obligate outcrossing, which together with inbreeding depression complicates traditional breeding approaches in obtaining plants with a stable growth habit. Inducing dominant mutations would provide an alternative strategy to introduce domestication traits in plants with high gene redundancy. Here we describe two complementary strategies to induce dominant mutations in the M. sativa genome and how they can be relevant in the control of flowering time. First, we outline a genome-engineering strategy that harnesses the use of microProteins as developmental regulators. MicroProteins are small proteins that appeared during genome evolution from genes encoding larger proteins. Genome-engineering allows us to retrace evolution and create microProtein-coding genes de novo. Second, we provide an inventory of genes regulated by microRNAs that control plant development. Making respective gene transcripts microRNA-resistant by inducing point mutations can uncouple microRNA regulation. Finally, we investigated the recently published genomes of M. sativa and provide an inventory of breeding targets, some of which, when mutated, are likely to result in dominant traits.

A microProtein repressor complex in the shoot meristem controls the transition to flowering.

MicroProteins are potent post-translational regulators. In Arabidopsis (Arabidopsis thaliana), the miP1a/b microProteins delay floral transition by forming a complex with CONSTANS (CO) and the co-repressor protein TOPLESS. To better understand the function of the miP1a microProtein in floral repression, we performed a genetic suppressor screen to identify suppressors of miP1a (sum) function. One mutant, sum1, exhibited strong suppression of the miP1a-induced late-flowering phenotype. Mapping of sum1 identified another allele of the gene encoding the histone H3K4 demethylase JUMONJI14 (JMJ14), which is required for miP1a function. Plants carrying mutations in JMJ14 exhibit an early flowering phenotype that is largely dependent on CO activity, supporting an additional role for CO in the repressive complex. We further investigated whether miP1a function involves chromatin modification, performed whole-genome methylome sequencing studies with plants ectopically expressing miP1a, and identified differentially methylated regions (DMRs). Among these DMRs is the promoter of FLOWERING LOCUS T (FT), the prime target of miP1a that is ectopically methylated in a JMJ14-dependent manner. Moreover, when aberrantly expressed at the shoot apex, CO induces early flowering, but only when JMJ14 is mutated. Detailed analysis of the genetic interaction among CO, JMJ14, miP1a/b, and TPL revealed a potential role for CO as a repressor of flowering in the shoot apical meristem (SAM). Altogether, our results suggest that a repressor complex operates in the SAM, likely to maintain it in an undifferentiated state until leaf-derived florigen signals induce SAM conversion into a floral meristem.

Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling.

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of LNJ in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

Global Analysis of Cereal microProteins Suggests Diverse Roles in Crop Development and Environmental Adaptation.

MicroProteins are a class of small single-domain proteins that post-translationally regulate larger multidomain proteins from which they evolved or which they relate to. They disrupt the normal function of their targets by forming microProtein-target heterodimers through compatible protein-protein interaction (PPI) domains. Recent studies confirm the significance of microProteins in the fine-tuning of plant developmental processes such as shoot apical meristem maintenance and flowering time regulation. While there are a number of well-characterized microProteins in Arabidopsis thaliana, studies from more complex plant genomes are still missing. We have previously developed miPFinder, a software for identifying microProteins from annotated genomes. Here we present an improved version where we have updated the algorithm to increase its accuracy and speed, and used it to analyze five cereal crop genomes - wheat, rice, barley, maize and sorghum. We found 20,064 potential microProteins from a total of 258,029 proteins in these five organisms, of which approximately 2000 are high-confidence, i.e., likely to function as actual microProteins. Gene ontology analysis of these 2000 microProtein candidates revealed their roles in stress, light and growth responses, hormone signaling and transcriptional regulation. Using a recently developed rice gene co-expression database, we analyzed 347 potential rice microProteins that are also conserved in other cereal crops and found over 50 of these rice microProteins to be co-regulated with their identified interaction partners. Overall, our study reveals a rich source of biotechnologically interesting small proteins that regulate fundamental plant processes such a growth and stress response that could be utilized in crop bioengineering.

Roadmap for Accelerated Domestication of an Emerging Perennial Grain Crop.

Shifting the life cycle of grain crops from annual to perennial would usher in a new era of agriculture that is more environmentally friendly, resilient to climate change, and capable of soil carbon sequestration. Despite decades of work, transforming the annual grain crop wheat (Triticum aestivum) into a perennial has yet to be realized. Direct domestication of wild perennial grass relatives of wheat, such as Thinopyrum intermedium, is an alternative approach. Here we highlight protein coding sequences in the recently released T. intermedium genome sequence that may be orthologous to domestication genes identified in annual grain crops. Their presence suggests a roadmap for the accelerated domestication of this plant using new breeding technologies.

Multi-level analysis of the interactions between REVOLUTA and MORE AXILLARY BRANCHES 2 in controlling plant development reveals parallel, independent and antagonistic functions.

Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play fundamental roles in controlling plant development. The known HD-ZIPIII target genes encode proteins involved in the production and dissipation of the auxin signal, HD-ZIPII transcription factors and components that feedback to regulate HD-ZIPIII expression or protein activity. Here, we have investigated the regulatory hierarchies of the control of MORE AXILLARY BRANCHES2 (MAX2) by the HD-ZIPIII protein REVOLUTA (REV). We found that REV can interact with the promoter of MAX2 In agreement, rev10D gain-of-function mutants had increased levels of MAX2 expression, while rev loss-of-function mutants showed lower levels of MAX2 in some tissues. Like REV, MAX2 plays known roles in the control of plant architecture, photobiology and senescence, which prompted us to initiate a multi-level analysis of growth phenotypes of hd-zipIII, max2 and respective higher order mutants thereof. Our data suggest a complex relationship of synergistic and antagonistic activities between REV and MAX2; these interactions appear to depend on the developmental context and do not all involve the direct regulation of MAX2 by REV.

Light affects tissue patterning of the hypocotyl in the shade-avoidance response.

Plants have evolved strategies to avoid shade and optimize the capture of sunlight. While some species are tolerant to shade, plants such as Arabidopsis thaliana are shade-intolerant and induce elongation of their hypocotyl to outcompete neighboring plants. We report the identification of a developmental module acting downstream of shade perception controlling vascular patterning. We show that Arabidopsis plants react to shade by increasing the number and types of water-conducting tracheary elements in the vascular cylinder to maintain vascular density constant. Mutations in genes affecting vascular patterning impair the production of additional xylem and also show defects in the shade-induced hypocotyl elongation response. Comparative analysis of the shade-induced transcriptomes revealed differences between wild type and vascular patterning mutants and it appears that the latter mutants fail to induce sets of genes encoding biosynthetic and cell wall modifying enzymes. Our results thus set the stage for a deeper understanding of how growth and patterning are coordinated in a dynamic environment.

Control of flowering in rice through synthetic microProteins.

Photoperiod-dependent flowering in rice is regulated by HEADING DATE 1 (Hd1), which acts as both an activator and repressor of flowering in a daylength-dependent manner. To investigate the use of microProteins as a tool to modify rice sensitivity to the photoperiod, we designed a synthetic Hd1 microProtein (Hd1miP) capable of interacting with Hd1 protein, and overexpressed it in rice. Transgenic OX-Hd1miP plants flowered significantly earlier than wild type plants when grown in non-inductive long day conditions. Our results show the potential of microProteins to serve as powerful tools for modulating crop traits and unraveling protein function.

Light Triggers the miRNA-Biogenetic Inconsistency for De-etiolated Seedling Survivability in Arabidopsis thaliana.

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

The B-Box-Containing MicroProtein miP1a/BBX31 Regulates Photomorphogenesis and UV-B Protection.

The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluence-dependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss- and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5 We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.

Spatiotemporal control of axillary meristem formation by interacting transcriptional regulators.

Branching is a common feature of plant development. In seed plants, axillary meristems (AMs) initiate in leaf axils to enable lateral shoot branching. AM initiation requires a high level of expression of the meristem marker SHOOT MERISTEMLESS (STM) in the leaf axil. Here, we show that modules of interacting transcriptional regulators control STM expression and AM initiation. Two redundant AP2-type transcription factors, DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL), control AM initiation by regulating STM expression. DRN and DRNL directly upregulate STM expression in leaf axil meristematic cells, as does another transcription factor, REVOLUTA (REV). The activation of STM expression by DRN/DRNL depends on REV, and vice versa. DRN/DRNL and REV have overlapping expression patterns and protein interactions in the leaf axil, which are required for the upregulation of STM expression. Furthermore, LITTLE ZIPPER3, another REV-interacting protein, is expressed in the leaf axil and interferes with the DRN/DRNL-REV interaction to negatively modulate STM expression. Our results support a model in which interacting transcriptional regulators fine-tune the expression of STM to precisely regulate AM initiation. Thus, shoot branching recruits the same conserved protein complexes used in embryogenesis and leaf polarity patterning.