Plant Developmental Transitions Research Articles

Interplay between coding and non-coding regulation drives the Arabidopsis seed-to-seedling transition

Translation of seed stored mRNAs is essential to trigger germination. However, when RNAPII re-engages RNA synthesis during the seed-to-seedling transition has remained in question. Combining csRNA-seq, ATAC-seq and smFISH in Arabidopsis thaliana we demonstrate that active transcription initiation is detectable during the entire germination process. Features of non-coding regulation such as dynamic changes in chromatin accessible regions, antisense transcription, as well as bidirectional non-coding promoters are widespread throughout the Arabidopsis genome. We show that sensitivity to exogenous ABSCISIC ACID (ABA) during germination depends on proximal promoter accessibility at ABA-responsive genes. Moreover, we provide genetic validation of the existence of divergent transcription in plants. Our results reveal that active enhancer elements are transcribed producing non-coding enhancer RNAs (eRNAs) as widely documented in metazoans. In sum, this study defining the extent and role of coding and non-coding transcription during key stages of germination expands our understanding of transcriptional mechanisms underlying plant developmental transitions.

The tomato EAR-motif repressor, SlERF36, accelerates growth transitions and reduces plant life cycle by regulating GA levels and responses.

Faster vegetative growth and early maturity/harvest reduce plant life cycle time and are important agricultural traits facilitating early crop rotation. GA is a key hormone governing developmental transitions that determine growth speed in plants. An EAR-motif repressor, SlERF36 that regulates various growth transitions, partly through regulation of the GA pathway and GA levels, was identified in tomato. Suppression of SlERF36 delayed germination, slowed down organ growth and delayed the onset of flowering time, fruit harvest and whole-plant senescence by 10-15 days. Its over-expression promoted faster growth by accelerating all these transitions besides increasing organ expansion and plant height substantially. The plant life cycle and fruit harvest were completed 20-30 days earlier than control without affecting yield, in glasshouse as well as net-house conditions, across seasons and generations. These changes in life cycle were associated with reciprocal changes in expression of GA pathway genes and basal GA levels between suppression and over-expression lines. SlERF36 interacted with the promoters of two GA2 oxidase genes, SlGA2ox3 and SlGA2ox4, and the DELLA gene, SlDELLA, reducing their transcription and causing a 3-5-fold increase in basal GA3/GA4 levels. Its suppression increased SlGA2ox3/4 transcript levels and reduced GA3/GA4 levels by 30%-50%. SlERF36 is conserved across families making it an important candidate in agricultural and horticultural crops for manipulation of plant growth and developmental transitions to reduce life cycles for faster harvest.

Untargeted Proteomics and Metabolomics Analysis of Plant Organ Development.

Our understanding of major developmental transitions in plants and animals has been transformed by the emergence of omics technologies. The majority of leaf growth research has been conducted at the transcriptional level. Although historically understudied, alterations at the protein and metabolite levels have begun to gain traction in recent years. Here, we present a protocol for metabolite and protein extraction followed by untargeted metabolomics and proteomics analysis of the growing leaves.

Past accomplishments and future challenges of the multi-omics characterization of leaf growth.

The advent of omics technologies has revolutionized biology and advanced our understanding of all biological processes, including major developmental transitions in plants and animals. Here, we review the vast knowledge accumulated concerning leaf growth in terms of transcriptional regulation before turning our attention to the historically less well-characterized alterations at the protein and metabolite level. We will then discuss how the advent of biochemical methods coupled with metabolomics and proteomics can provide insight into the protein–protein and protein–metabolite interactome of the growing leaves. We finally highlight the substantial challenges in detection, spatial resolution, integration, and functional validation of the omics results, focusing on metabolomics as a prerequisite for a comprehensive understanding of small-molecule regulation of plant growth.

Assessing Flowering Time Under Different Photoperiods.

Flowering time is one of the most important developmental transitions in plants, especially in annuals such as Arabidopsis thaliana. However, flowering is also a critical agronomic trait, as it impacts the level of vegetative biomass produced (e.g., leaves) or the amount of seed (grain) generated. Therefore, uncovering flowering phenotypes would help understand the impact of any regulatory network on the overall plant life cycle, since flowering integrates multiple cues, both environmental (e.g., photoperiod, temperature) and internal (e.g., induction/repression of specific genes, phytohormone accumulation, plant age). Although the photoperiod flowering pathway has been extensively studied, and its gene circuitry characterized in great detail, specific flowering time protocols are mostly accessible to specialized laboratories in this field. In this report, we address this knowledge gap by generating a reproducible, non-expensive, and step-by-step protocol to assess flowering time under different photoperiods. We provide a comprehensive description and highlight the major pitfalls in the process. Moreover, this protocol could be expanded to include temperature changes and thus contribute to assess the impact of both environmental conditions in the plant's decision to flower.

Beyond the Genetic Pathways, Flowering Regulation Complexity in Arabidopsis thaliana

Flowering is one of the most critical developmental transitions in plants’ life. The irreversible change from the vegetative to the reproductive stage is strictly controlled to ensure the progeny’s success. In Arabidopsis thaliana, seven flowering genetic pathways have been described under specific growth conditions. However, the evidence condensed here suggest that these pathways are tightly interconnected in a complex multilevel regulatory network. In this review, we pursue an integrative approach emphasizing the molecular interactions among the flowering regulatory network components. We also consider that the same regulatory network prevents or induces flowering phase change in response to internal cues modulated by environmental signals. In this sense, we describe how during the vegetative phase of development it is essential to prevent the expression of flowering promoting genes until they are required. Then, we mention flowering regulation under suboptimal growing temperatures, such as those in autumn and winter. We next expose the requirement of endogenous signals in flowering, and finally, the acceleration of this transition by long-day photoperiod and temperature rise signals allowing A. thaliana to bloom in spring and summer seasons. With this approach, we aim to provide an initial systemic view to help the reader integrate this complex developmental process.

Redundant and specific roles of individual MIR172 genes in plant development.

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

Molecular regulation of plant developmental transitions and plant architecture via PEPB family proteins: an update on mechanism of action.

This year marks the 100th anniversary of the experiments by Garner and Allard that showed that plants measure the duration of the night and day (the photoperiod) to time flowering. This discovery led to the identification of Flowering Locus T (FT) in Arabidopsis and Heading Date 3a (Hd3a) in rice as a mobile signal that promotes flowering in tissues distal to the site of cue perception. FT/Hd3a belong to the family of phosphatidylethanolamine-binding proteins (PEBPs). Collectively, these proteins control plant developmental transitions and plant architecture. Several excellent recent reviews have focused on the roles of PEBPs in diverse plant species; here we will primarily highlight recent advances that enhance our understanding of the mechanism of action of PEBPs and discuss critical open questions.

Boron: More Than an Essential Element for Land Plants?

Although boron (B) is an element that has long been assumed to be an essential plant micronutrient, this assumption has been recently questioned. Cumulative evidence has demonstrated that the players associated with B uptake and translocation by plant roots include a sophisticated set of proteins used to cope with B levels in the soil solution. Here, we summarize compelling evidence supporting the essential role of B in mediating plant developmental programs. Overall, most plant species studied to date have exhibited specific B transporters with tight genetic coordination in response to B levels in the soil. These transporters can uptake B from the soil, which is a highly uncommon occurrence for toxic elements. Moreover, the current tools available to determine B levels cannot precisely determine B translocation dynamics. We posit that B plays a key role in plant metabolic activities. Its importance in the regulation of development of the root and shoot meristem is associated with plant developmental phase transitions, which are crucial processes in the completion of their life cycle. We provide further evidence that plants need to acquire sufficient amounts of B while protecting themselves from its toxic effects. Thus, the development of in vitro and in vivo approaches is required to accurately determine B levels, and subsequently, to define unambiguously the function of B in terrestrial plants.

Auxin Regulates Sucrose Transport to Repress Petal Abscission in Rose (Rosa hybrida).

Developmental transitions in plants require adequate carbon resources, and organ abscission often occurs due to competition for carbohydrates/assimilates. Physiological studies have indicated that organ abscission may be activated by Suc deprivation; however, an underlying regulatory mechanism that links Suc transport to organ shedding has yet to be identified. Here, we report that transport of Suc and the phytohormone auxin to petals through the phloem of the abscission zone (AZ) decreases during petal abscission in rose (Rosa hybrida), and that auxin regulates Suc transport into the petals. Expression of the Suc transporter RhSUC2 decreased in the AZ during rose petal abscission. Similarly, silencing of RhSUC2 reduced the Suc content in the petals and promotes petal abscission. We established that the auxin signaling protein RhARF7 binds to the promoter of RhSUC2, and that silencing of RhARF7 reduces petal Suc contents and promotes petal abscission. Overexpression of RhSUC2 in the petal AZ restored accelerated petal abscission caused by RhARF7 silencing. Moreover, treatment of rose petals with auxin and Suc delayed ethylene-induced abscission, whereas silencing of RhARF7 and RhSUC2 accelerated ethylene-induced petal abscission. Our results demonstrate that auxin modulates Suc transport during petal abscission, and that this process is regulated by a RhARF7-RhSUC2 module in the AZ.

The Important Function of Mediator Complex in Controlling the Developmental Transitions in Plants.

Developmental transitions in plants are tightly associated with changes in the transcriptional regulation of gene expression. One of the most important regulations is conferred by cofactors of RNA polymerase II including the mediator complex, a large complex with a modular organization. The mediator complex recruits transcription factors to bind to the specific sites of genes including protein-coding genes and non-coding RNA genes to promote or repress the transcription initiation and elongation using a protein-protein interaction module. Mediator complex subunits have been isolated and identified in plants and the function of most mediator subunits in whole life cycle plants have been revealed. Studies have shown that the Mediator complex is indispensable for the regulation of plant developmental transitions by recruiting age-, flowering-, or hormone-related transcription factors. Here, we first overviewed the Mediator subunits in plants, and then we summarized the specific Mediator subunits involved in developmental transitions, including vegetative phase change and floral transition. Finally, we proposed the future directions to further explore their roles in plants. The link between Mediator subunits and developmental transitions implies the necessity to explore targets of this complex as a potential application in developing high quality crop varieties.

The Control of Developmental Phase Transitions by microRNAs and Their Targets in Seed Plants.

Seed plants usually undergo various developmental phase transitions throughout their lifespan, mainly including juvenile-to-adult and vegetative-to-reproductive transitions, as well as developmental transitions within organ/tissue formation. MicroRNAs (miRNAs), as a class of small endogenous non-coding RNAs, are involved in the developmental phase transitions in plants by negatively regulating the expression of their target genes at the post-transcriptional level. In recent years, cumulative evidence has revealed that five miRNAs, miR156, miR159, miR166, miR172, and miR396, are key regulators of developmental phase transitions in plants. In this review, the advanced progress of the five miRNAs and their targets in regulating plant developmental transitions, especially in storage organ formation, are summarized and discussed, combining our own findings with the literature. In general, the functions of the five miRNAs and their targets are relatively conserved, but their functional divergences also emerge to some extent. In addition, potential research directions of miRNAs in regulating plant developmental phase transitions are prospected.

Cloning, subcellular localization, and expression analysis of VvRING1a from grapevine

Polycomb group (PcG) proteins within polycomb repressive complex 1 (PRC1) and PRC2 are significant epigenetic regulation factors involved in controlling plant developmental transitions. PRC2 catalyzes H3K27 trimethylation (H3K27me3), and PRC1 recognizes the site of H3K27me3 and catalyzes H2AK119 monoubiquitination to maintain transcription repression. PRC2 components are conserved from plants to animals and have been investigated for a long time. However, the compositions and functions of PRC1 vary between animals and plants. Plant PRC1 has only been confirmed in recent years. Very little research has been conducted on PcG in grapevine (Vitis vinifera ‘Cabernet Sauvignon’). Here, we cloned the full-length coding sequence of the VvRING1a gene, which is homologous with the Arabidopsis thaliana PRC1 core component AtRING1a. We then analyzed the structure, subcellular localization, and expression pattern. The tissue-specific expression analysis showed that VvRING1a is expressed in various tissues, including in roots, stems, leaves, flowers, buds, and fruits. This study will be a useful reference for further study of VvRING1a.

PIF transcription factors link a neighbor threat cue to accelerated reproduction in Arabidopsis

Changes in light quality indicative of competition for this essential resource influence plant growth and developmental transitions; however, little is known about neighbor proximity-induced acceleration of reproduction. Phytochrome B (phyB) senses light cues from plant competitors, ultimately leading to the expression of the floral inducers FLOWERING LOCUS T (FT) and TWIN SISTER of FT (TSF). Here we show that PHYTOCHROME INTERACTING FACTORs 4, 5 and 7 (PIF4, PIF5 and PIF7) mediate neighbor proximity-induced flowering, with PIF7 playing a prominent role. These transcriptional regulators act directly downstream of phyB to promote expression of FT and TSF. Neighbor proximity enhances PIF accumulation towards the end of the day, coinciding with enhanced floral inducer expression. We present evidence supporting direct PIF-regulated TSF expression. The relevance of our findings is illustrated by the prior identification of FT, TSF and PIF4 as loci underlying flowering time regulation in natural conditions.

PcG Proteins MSI1 and BMI1 Function Upstream of miR156 to Regulate Aerial Tuber Formation in Potato.

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member, represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon, whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects, including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhanced miR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further, grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increased accumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover, we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax group H3K4-methyl-transferases in StMSI1-OE Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor.

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first Förster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

DNA Methylation Analysis of Dormancy Release in Almond (Prunus dulcis) Flower Buds Using Epi-Genotyping by Sequencing.

DNA methylation and histone post-translational modifications have been described as epigenetic regulation mechanisms involved in developmental transitions in plants, including seasonal changes in fruit trees. In species like almond (Prunus dulcis (Mill.) D.A: Webb), prolonged exposure to cold temperatures is required for dormancy release and flowering. Aiming to identify genomic regions with differential methylation states in response to chill accumulation, we carried out Illumina reduced-representation genome sequencing on bisulfite-treated DNA from floral buds. To do this, we analyzed almond genotypes with different chilling requirements and flowering times both before and after dormancy release for two consecutive years. The study was performed using epi-Genotyping by Sequencing (epi-GBS). A total of 7317 fragments were sequenced and the samples compared. Out of these fragments, 677 were identified as differentially methylated between the almond genotypes. Mapping these fragments using the Prunus persica (L.) Batsch v.2 genome as reference provided information about coding regions linked to early and late flowering methylation markers. Additionally, the methylation state of ten gene-coding sequences was found to be linked to the dormancy release process.

Feedback regulation of COOLAIR expression controls seed dormancy and flowering time.

Plants integrate seasonal signals, including temperature and day length, to optimize the timing of developmental transitions. Seasonal sensing requires the activity of two proteins, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT), that control certain developmental transitions in plants. During reproductive development, the mother plant uses FLC and FT to modulate progeny seed dormancy in response to temperature. We found that for regulation of seed dormancy, FLC and FT function in opposite configuration to how those same genes control time to flowering. For seed dormancy, FT regulates seed dormancy through FLC gene expression and regulates chromatin state by activating antisense FLC transcription. Thus, in Arabidopsis the same genes controlled in opposite format regulate flowering time and seed dormancy in response to the temperature changes that characterize seasons.

The autonomous flowering-time pathway pleiotropically regulates seed germination in Arabidopsis thaliana.

Two critical developmental transitions in plants are seed germination and flowering, and the timing of these transitions has strong fitness consequences. How genetically independent the regulation of these transitions is can influence the expression of life cycles. This study tested whether genes in the autonomous flowering-time pathway pleiotropically regulate flowering time and seed germination in the genetic model Arabidopsis thaliana, and tested whether the interactions among those genes are concordant between flowering and germination stages. Several autonomous-pathway genes promote flowering and impede germination. Moreover, the interactions among those genes were highly concordant between the regulation of flowering and germination. Despite some degree of functional divergence between the regulation of flowering and germination by autonomous-pathway genes, the autonomous pathway is highly functionally conserved across life stages. Therefore, genes in the autonomous flowering-time pathway are likely to contribute to genetic correlations between flowering and seed germination, possibly contributing to the winter-annual life history.

Oxygen, Energy, and Light Signalling Direct Meristem Fate.

Light, nutrition, and oxygen are each important cues for developmental transitions in plants. A small number of recent studies have converged to give the first indication of how these environmental cues act together and independently via auxin, cytokinin, and ethylene to regulate cell proliferation in the root and shoot meristems.