Phloem Development Research Articles

PagNAC2a promotes phloem fiber development by regulating PagATL2 in poplar

Phloem fiber is a key component of phloem tissue and is involved in supporting its structural integrity. NAC domain transcription factors are master switches that regulate secondary cell wall (SCW) biosynthesis in xylem fibers, but the mechanism by which NACs regulate phloem fiber development remains unexplored. Here, a NAC2-like gene in poplar, PagNAC2a, was shown to be involved in phloem fiber differentiation. qRT-PCR and GUS staining revealed that PagNAC2a was specifically expressed in the phloem zone of poplar stems. The overexpression of PagNAC2a in poplar increased plant biomass by increasing plant height, stem diameter, and leaf area. Stem anatomy analysis revealed that overexpression of PagNAC2a resulted in enhanced phloem fiber differentiation and cell wall deposition. In addition, PagNAC2a directly upregulated the expression of PagATL2, a gene involved in phloem development, as revealed by yeast one hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) assays. Overall, we proposed that the PagNAC2a was a positive regulator of phloem fiber development in poplar, and these results provided insights into the molecular mechanisms involved in the differentiation of phloem fibers.

Laser wounding pattern in relation to vascular tissue development for the stimulation of adventitious root formation in rose cuttings

The stimulation of adventitious root formation from laser-wounded rose cuttings in our previous study suggests that exposing the phloem proximities is one of the most relevant aspects for a positive effect on rooting response. But, the specific dimensions that wound patterns must fulfill to optimize rooting promotion remain unknown. This study analyzed the effect of wounded area and wound perimeter of laser marking patterns on the development of phloem, xylem, and callus using cross sections of single-leaf cuttings of Rosa canina 'Pfänder'. Four distinct laser patterns were designed and marked along the cutting base. Among these, three patterns were based on longitudinal strips, while one pattern was characterized by small squares, resulting in two distinct wound area levels and four wound perimeter levels. Periodic evaluations of stem sections showed that the development of phloem and xylem was significantly influenced by the pattern's geometry. Larger dimensions of xylem were associated with patterns of greater area and a smaller perimeter, while an increase in phloem was related to patterns of longer perimeter distributed in smaller areas. The maximum rooting success in wounded cuttings reached 44% in contrast to 9% observed in the control group in the absence of additional wounds. The development of vascular tissue was significantly correlated with adventitious rooting, with phloem being more closely linked with a Pearson coefficient of 0.92 compared to 0.30 for xylem. Additionally, a negative Pearson coefficient of −0.92 between the ratio area: perimeter and adventitious root formation showed that laser patterns with large wounded area with less borders led to a reduced rooting response. The results provide evidence of how wounded tissue contributes to the intrinsic development of adventitious roots and reveal the importance of proper wound dimensions.

Transcriptome sequencing-based analysis of primary vein development in Betula pendula ‘Dalecarlica’

Keymessage The study revealed the major biological processes occurred at three developmental stages and identified candidate genes involved in primary vein development of birch plants.Vascular tissues usually mirror the surrounding leaf shape and its development plays a fundamental role in plant performance. However, the information of vascular development in birch trees, especially primary vein development, remains unclear. Therefore, we conducted the anatomical observation on primary veins from leaves at different development stages in Betula pendula ‘Dalecarlica’. With the development of primary vein, dynamic changes in mechanical tissue thickness and primary vein diameter were consistent with each other, and the sum of phloem, xylem and cambium thickness was significantly varied. Transcriptome analysis indicated that primary vein development could be divided into three stages, namely Stage I, II and III, which were in aggreement with anatomical observation. Expression of marker genes associated with vascular tissues revealed that pro-vasculature development occurred at Stage I and II, and phloem development occurred at Stage III. GO enrichment analysis of differentially expressed genes (DEGs) showed that shared DEGs at Stage II were mainly engaged in cell division and cell cycle, and shared DEGs at Stage III were mainly engaged in phosphorylation. Decreased cell division and cell cycle as well as activation of lignin biosynthesis might contribute to primary vein development. Combining phenotypic traits, we performed weighted gene co-expression network analysis and identified a cytochrome P450 84A (CYP84A) family gene (BpF5H1). Based on analyses of gene families, expression patterns and yeast-two hybrid assay results, we proposed a potential electron transfer pathway involving BpF5H1 and three cytochrome b5 proteins during primary vein development in B. pendula ‘Dalecarlica’. These results could shed some light on which biological processes occurred during primary vein formation and provide some valuable clues for vascular morphogenesis in woody plants.

A multispecies study reveals the diversity and potential regulatory role of long noncoding RNAs in cucurbits.

Plant long noncoding RNAs (lncRNAs) exhibit features such as tissue-specific expression, spatiotemporal regulation, and stress responsiveness. Although diverse studies support the regulatory role of lncRNAs in model plants, our knowledge about lncRNAs in crops is limited. We employ a custom pipeline on a dataset of over 1000 RNA-seq samples across nine representative species of the family Cucurbitaceae to predict 91 209 nonredundant lncRNAs. The lncRNAs were characterized according to three confidence levels and classified by their genomic context into intergenic, natural antisense, intronic, and sense-overlapping. Compared with protein-coding genes, lncRNAs were, on average, expressed at low levels and displayed significantly higher specificity when considering tissue, developmental stages, and stress responsiveness. The evolutionary analysis indicates higher positional conservation than sequence conservation, probably linked to the conserved modular motifs within syntenic lncRNAs. Moreover, a positive correlation between the expression of intergenic/natural antisense lncRNAs and their closest/parental gene was observed. For those intergenic, the correlation decreases with the distance to the neighboring gene, supporting that their potential cis-regulatory effect is within a short-range. Furthermore, the analysis of developmental studies showed that a conserved NAT-lncRNA family is differentially expressed in a coordinated way with their cognate sense protein-coding genes. These genes code for proteins associated with phloem development, thus providing insights about the potential involvement of some of the identified lncRNAs in a developmental process. We expect that this extensive inventory will constitute a valuable resource for further research lines focused on elucidating the regulatory mechanisms mediated by lncRNAs in cucurbits.

Ontogeny of the multiple cambial variants and secondary growth in some species of Merremia and related genera (Convolvulaceae)

ABSTRACT The present study investigates the stem anatomy and development of cambial variants in 10 species of Merremia and related genera (Convolvulaceae) were compared histologically. Among them, Camonea kingii, C. umbellata, Distimake aegyptius, D. quinquefolius, D. vitifolius and M. hederacea increased their stem diameter by initiating successive cambia. In contrast, remaining species showed regular secondary growth. These species also demonstrated inversely oriented vascular elements, phloem wedges, intraxylary cambia, parenchyma proliferation and development of radial vascular cylinders in the large rays of xylem. Phloem wedges developed by the differential activity of regular cambium in small segments by depositing only unlignified parenchyma internally whereas nearby sectors of the cambium behaved regularly. After the development of intraxylary protophloem from procambial derivatives, additional phloem was formed from the pith cells and by initiating intraxylary cambium. Initially, intraxylary cambium was unifacial in all species, but eventually became bifacial (except M. emarginata) and deposited both xylem and phloem. In conclusion, Merremia species use all feasible methods to generate new vascular elements to compensate for the supply of water, nutrients and photosynthate. Inter-and intraxylary phloem development appears to provide an additional and safe conduit for long-distance vertical conduction, whereas radial vascular cylinders facilitate radial conduction.

Physiological and developmental disturbances caused by Botryosphaeria dieback in the annual stems of grapevine.

Botryosphaeria dieback is a grapevine trunk disease caused by fungi of the Botryosphaeriaceae family, which attacks more specifically the woody tissues. The infection leads to different symptoms including a severe form with a leaf drop as well as premature plant death. Botryosphaeria dieback causes major economic losses, since no effective treatment is yet available. A better understanding is necessary to find solutions to fight this disease. In this study, our objective was to characterize the "leaf drop" form by (1) looking for the presence of pathogens in the basal internodes of stems, (2) quantifying blocked vessels by tylosis and/or gummosis, and (3) describing the impact of the disease on vine physiology (gene expression and metabolome) and development (establishment and functioning of the cambium and phellogen) at the level of xylem and phloem of basal stem internodes. Our study has shown that Botryosphaeriaceae were present in both phloem and xylem of the basal internodes of the annual stem, with xylem vessels obturated. We have also clearly demonstrated that gene expression and metabolite profiles were strongly modified in both xylem and phloem of diseased plants. Differences in stems between healthy (control, C) and diseased (D) plants were low at flowering (vines not yet symptomatic), higher at the onset of symptom expression and still present, although less marked, at full disease expression. qRT-PCR analysis showed in both phloem and xylem an overexpression of genes involved in plant defense, and a repression of genes related to meristematic activity (i.e. vascular cambium and phellogen). Metabolomic analysis showed specific fingerprints in stems of healthy and diseased plants from the onset of symptom expression, with an increase of the level of phytoalexins and mannitol, and a decrease of 1-kestose one. At the structural level, many alterations were observed in internodes, even before the onset of symptoms: a classical obstruction of xylem vessels and, for the first time, a disorganization of the secondary phloem with an obstruction of the sieve plates by callose. The disease modifies the development of both secondary phloem (liber) and phellogen. Altogether, this study combining different approaches allowed to highlight deep vine dysfunction in the internodes at the base of stems, that may explain vine decline due to Botryosphaeria dieback.

Temporal and spatial variability of phloem structure in Picea abies and Fagus sylvatica and its link to climate.

Using a unique 8-year data set (2010-2017) of phloem data, we studied the effect of temperature and precipitation on the phloem anatomy (conduit area, widths of ring, early and late phloem) and xylem-ring width in two coexisting temperate tree species, Picea abies and Fagus sylvatica, from three contrasting European temperate forest sites. Histometric analyses were performed on microcores taken from tree stems in autumn. We found high interannual variability and sensitivity of phloem anatomy and xylem-ring widths to precipitation and temperature; however, the responses were species- and site-specific. The contrasting response of xylem and phloem-ring widths of the same tree species to weather conditions was found at the two Slovenian sites generally well supplied with precipitation, while at the driest Czech site, the influence of weather factors on xylem and phloem ring widths was synchronised. Since widths of mean annual xylem and phloem increments were narrowest at the Czech site, this site is suggested to be most restrictive for the radial growth of both species. By influencing the seasonal patterns of xylem and phloem development, water availability appears to be the most important determinant of tissue- and species-specific responses to local weather conditions.

Polyploid Genome Assembly Provides Insights into Morphological Development and Ascorbic Acid Accumulation of Sauropus androgynus.

Sauropus androgynus (S. androgynus) (2n = 4x = 52) is one of the most popular functional leafy vegetables in South and Southeast Asia. With its rich nutritional and pharmaceutical values, it has traditionally had widespread use for dietary and herbal purposes. Here, the genome of S. androgynus was sequenced and assembled, revealing a genome size of 1.55 Gb with 26 pseudo-chromosomes. Phylogenetic analysis traced back the divergence of Sauropus from Phyllanthus to approximately 29.67 million years ago (Mya). Genome analysis revealed that S. androgynus polyploidized around 20.51 Mya and shared a γ event about 132.95 Mya. Gene function analysis suggested that the expansion of pathways related to phloem development, lignin biosynthesis, and photosynthesis tended to result in the morphological differences among species within the Phyllanthaceae family, characterized by varying ploidy levels. The high accumulation of ascorbic acid in S. androgynus was attributed to the high expression of genes associated with the L-galactose pathway and recycling pathway. Moreover, the expanded gene families of S. androgynus exhibited multiple biochemical pathways associated with its comprehensive pharmacological activity, geographic adaptation and distinctive pleasurable flavor. Altogether, our findings represent a crucial genomic asset for S. androgynus, casting light on the intricate ploidy within the Phyllanthaceae family.

Ontogeny of multiple variants in the stems of Phaseolus lunatus L. (Fabaceae)

Development of the cambial variant is characteristic to most of the climbing members of the Fabaceae and is considered as an alternative path to increase their stem diameter for efficient supply of minerals and photosynthate within limited stem diameter. These alternative forms of secondary growths are ascribed as an adaptive character to increase stem flexibility and most often they are found to be taxon-specific. In the present study, histological investigation on the main stems of Phaseolus lunatus L. (Fabaceae) showed multiple combinations of variant secondary growth like the formation of vascular cylinders external to the secondary phloem and proliferation of parenchyma (axial and ray) followed by the development of interxylary phloem, interxylary cambia (functionally regular and inverse) and ray cambia. The secondary xylem produced by regular cambium was diffuse-porous with indistinct growth rings and composed of an abundance of thin-walled unlignified parenchyma. With the increase in age, phloem rays underwent dilatation, subsequently became meristematic and formed oval to circular vascular cylinders/units with irregular orientation. Concomitantly, dedifferentiation xylem parenchyma not only formed small islands of interxylary phloem but also led to the origin of interxylary cambial segments that differentiated bidirectionally and deposited xylem and phloem having inverse, diagonal, tangential or radial orientations. Similar cell division activity was also observed in marginal xylem ray cells (ray cambium) that produced both the xylem and phloem on either side. All these cambial variants are described in detail and their possible significance in the climbing habit is discussed.

Development, differentiation, and material distribution of secondary phloem in Pinus massoniana

Development, differentiation, and material distribution of secondary phloem in Pinus massoniana

Phloem development.

The evolution of the plant vascular system is a key process in Earth history because it enabled plants to conquer land and transform the terrestrial surface. Among the vascular tissues, the phloem is particularly intriguing because of its complex functionality. In angiosperms, its principal components are the sieve elements, which transport phloem sap, and their neighboring companion cells. Together, they form a functional unit that sustains sap loading, transport, and unloading. The developmental trajectory of sieve elements is unique among plant cell types because it entails selective organelle degradation including enucleation. Meticulous analyses of primary, so-called protophloem in the Arabidopsis thaliana root meristem have revealed key steps in protophloem sieve element formation at single-cell resolution. A transcription factor cascade connects specification with differentiation and also orchestrates phloem pole patterning via noncell-autonomous action of sieve element-derived effectors. Reminiscent of vascular tissue patterning in secondary growth, these involve receptor kinase pathways, whose antagonists guide the progression of sieve element differentiation. Receptor kinase pathways may also safeguard phloem formation by maintaining the developmental plasticity of neighboring cell files. Our current understanding of protophloem development in the A. thaliana root has reached sufficient detail to instruct molecular-level investigation of phloem formation in other organs.

Formation of inter-and intraxylary phloem in some species of Argyreia Lour. (Convolvulaceae)

The development of inter-and intraxylary phloem was investigated in nineteen species of Argyreia Lour. Among all species, differentiation of intraxylary protophloem was observed concomitant to the regular external protophloem from the procambial derivatives (except A. nervosa). However, A. nervosa, showed the formation of medullary bundles along with the regular protoxylem while the development of intraxylary phloem was observed only after the establishment of a complete ring of the vascular cambium and deposition of a few secondary xylem elements. In all the species (except A. nervosa), additional intraxylary phloem differentiated from the adjacent parenchyma and the intraxylary cambium initiated at the periphery of the pith. It divided uni-directionally and showed deposition of only phloem elements. In the transverse view, sieve elements of intraxylary phloem were oval to circular, rectangular, or multi-angled or sometimes in a cluster of three to several cells with simple and compound sieve plates oriented obliquely on their terminal ends. In the thick stems of A. cuneata, A. elliptica, A. lawii, A. mastersii, A. nervosa, A. sericea, A. sharadchandrajii and A. splendens, thin-walled unlignified axial parenchyma strands embedded within the secondary xylem divide and differentiated into isolated or groups of interxylary phloem elements. In the transverse view, these sieve elements were like regular phloem (oval to circular) in appearance but occasionally they may be narrow in diameter, tangentially flattened and arranged in clusters. In thick stems, earlier-formed sieve elements of the interxylary phloem collapse and get crushed due to the expansion of adjacent cells. The remaining species though showed the presence of parenchyma strands within the secondary xylem, no interxylary phloem was observed even in thick stems. Such parenchyma cells within islands always showed the presence of druses.

OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module driving phloem development

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

Structure and development of phloem in some species of Merremia Dennst. ex Endl. sensu lato (Convolvulaceae)

Convolvulaceae is known for the occurrence of cambial variants but studies on phloem development are rare. In the present study, the structure and development of primary and secondary phloem in seven species of Merremia Dennst. ex Endl. s.l. (Convolvulaceae) were investigated. All the examined species were characterized by the presence of inter‐ and intraxylary phloem, except M. emarginata (Burm.f.) Hallier f. which showed an absence of interxylary phloem. The secondary xylem of all the species was characterized by the presence of phloem wedges and thin‐walled parenchyma strands/islands. Among them, all the species showed the formation of interxylary phloem from unlignified parenchyma cells located in the phloem wedges, except Distimake dissectus (Jacq.) A.R.Simões & Staples (syn: M. dissecta (Jacq.) Hallier f.) and M. emarginata. Its development was also observed in the parenchyma strands/pockets of M. hederacea (Jacq.) Hallier f. and Camonea vitifolia (Burm.f.) A.R.Simões & Staples (syn: M. vitifolia (Jacq.) Hallier f.), while in the rest of the species such pockets were present but no interxylary phloem was observed in them. In relatively thick stems, a quantifiable amount of intraxylary secondary phloem is accumulated by the activity of the intraxylary phloem (internal) cambium. The structure and dimensions of the sieve tube elements significantly vary in all three different types of phloem (i.e. regular external, inter‐ and intraxylary). Sieve elements may possess simple or compound sieve plates and may be wide or narrow in diameter within the same species. The presence of inter‐ and intraxylary phloem and phloem wedges is a characteristic feature of all the investigated species while the absence of interxylary phloem in M. emarginata may be associated with its habit as it spreads on the ground. More studies are needed on species that show the presence of wide and narrow sieve elements, which may contribute to understating the functional ecology and evolution of climbing habits. Further studies are recommended on thick stems of all the investigated species along with other species of Merremia to understand their taxonomic position because some of them were recently transferred to other genera.

Lelliottia nimipressuralis (Carter 1945) Brady et al. 2013 as the causative agent of bacterial wetwood disease of common silver fir (Abies alba mill.)

Abstract In recent decades, in many regions of the planet, there has been a widespread deterioration in the health condition and dieback of dark coniferous forests, caused by the combined action of various stress factors of biotic and abiotic origin. Forests with the participation of species of the genus Abies Mill. are particularly prone to degradation and dieback. The aim of the research is to study the symptoms of bacterial wetwood disease of Abies alba in the Ivano-Frankivsk region (state enterprise ‘Kutske forestry’) and to determine the anatomical, morphological and cultural properties of the pathogen. Some of the common symptoms of the disease include cracks and ulcers on the trunks with exfoliated rhytidome and exudate secretion, massive development of epicormic shoots, saturated xylem and phloem, wet rot with a characteristic odour of fermentation and pathological nucleus. Based on the syntaxonomic analysis of fir forests, an attempt has been made to identify the objective causes that lead to excessive development of phytopathogens and dieback of Abies alba. Our research established that the primary cause of dieback of Abies alba Mill. forests is a systemic, vascular–parenchymal disease known as bacterial wetwood of fir, which affects all plant tissues at all stages of ontogenesis. We isolated and experimentally confirmed that the causative agent of the disease is a phytopathogenic bacterium Lelliottia nimipressuralis (Carter 1945) Brady et al. 2013, which also causes bacterial wetwood in many species of forest woody plants, and also studied its common morphological, physiological and biochemical properties.

A Dof-CLE circuit controls phloem organization.

The phloem consists of sieve elements (SEs) and companion cells (CCs). Here we show that Dof-class transcription factors preferentially expressed in the phloem (phloem-Dofs) are not only necessary and sufficient for SE and CC differentiation, but also induce negative regulators of phloem development, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED25 (CLE25), CLE26 and CLE45 secretory peptides. CLEs were perceived by BARELY ANY MERISTEM (BAM)-class receptors and CLAVATA3 INSENSITIVE RECEPTOR KINASE (CIK) co-receptors, and post-transcriptionally decreased phloem-Dof proteins and repressed SE and CC formation. Multiple mutations in CLE-, BAM- or CIK-class genes caused ectopic formation of SEs and CCs, producing an SE/CC cluster at each phloem region. We propose that while phloem-Dofs induce phloem cell formation, they inhibit excess phloem cell formation by inducing CLEs. Normal-positioned SE and CC precursor cells appear to overcome the effect of CLEs by reinforcing the production of phloem-Dofs through a positive feedback transcriptional regulation.

OsAPL controls the nutrient transport systems in the leaf of rice (Oryza sativa L.).

OsAPL positively controls the seedling growth and grain size in rice by targeting the plasma membrane H+-ATPase-encoding gene, OsRHA1, as well as drastically affects genes encoding H+-coupled secondary active transporters. Nutrient transport is a key component of both plant growth and environmental adaptation. Photosynthates and nutrients produced in the source organs (e.g., leaves) need to be transported to the sink organs (e.g., seeds). In rice, the unloading of nutrients occurs through apoplastic transport (i.e., across the membrane via transporters) and is dependent on the efficiency and number of transporters embedded in the cell membrane. However, the genetic mechanisms underlying the regulation of these transporters remain to be determined. Here we show that rice (Oryza sativa L., Kitaake) ALTERED PHLOEM DEVELOPMENT (OsAPL), homologous to a MYB family transcription factor promoting phloem development in Arabidopsis thaliana, regulates the number of transporters in rice. Overexpression of OsAPL leads to a 10% increase in grain yield at the heading stage. OsAPL acts as a transcriptional activator of OsRHA1, which encodes a subunit of the plasma membrane H+-ATPase (primary transporter). In addition, OsAPL strongly affects the expression of genes encoding H+-coupled secondary active transporters. Decreased expression of OsAPL leads to a decreased expression level of nutrient transporter genes. Taken together, our findings suggest the involvement of OsAPL in nutrients transport and crop yield accumulation in rice.

JULGI-mediated increment in phloem transport capacity relates to fruit yield in tomato.

SummaryThe continuous growth of the global population and the increase in the amount of arid land has severely constrained agricultural crop production. To solve this problem, many researchers have attempted to increase productivity through the efficient distribution of energy; however, the direct relationship between the plant vasculature, specifically phloem development, and crop yield is not well established. Here, we demonstrate that an optimum increase in phloem‐transportation capacity by reducing SIJUL expression leads to improved sink strength in tomato (Solanum lycopersicum L.). SIJUL, a negative regulator of phloem development, suppresses the translation of a positive regulator of phloem development, SlSMXL5. The suppression of SlJUL increases the number of phloem cells and sucrose transport, but only an optimal reduction of SlJUL function greatly enhances sink strength in tomato, improving fruit setting, and yield contents by 37% and 60%, respectively. We show that the increment in phloem cell number confers spare transport capacity. Our results suggest that the control of phloem‐transport capacity within the threshold could enhance the commitment of photosynthates to instigate yield improvement.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

The Candidate Photoperiod Gene MtFE Promotes Growth and Flowering in Medicago truncatula

Flowering time influences the yield and productivity of legume crops. Medicago truncatula is a reference temperate legume that, like the winter annual Arabidopsis thaliana, shows accelerated flowering in response to vernalization (extended cold) and long-day (LD) photoperiods (VLD). However, unlike A. thaliana, M. truncatula appears to lack functional homologs of core flowering time regulators CONSTANS (CO) and FLOWERING LOCUS C (FLC) which act upstream of the mobile florigen FLOWERING LOCUS T (FT). Medicago truncatula has three LD-induced FT-like genes (MtFTa1, MtFTb1, and MtFTb2) with MtFTa1 promoting M. truncatula flowering in response to VLD. Another photoperiodic regulator in A. thaliana, FE, acts to induce FT expression. It also regulates the FT transport pathway and is required for phloem development. Our study identifies a M. truncatula FE homolog Medtr6g444980 (MtFE) which complements the late flowering fe-1 mutant when expressed from the phloem-specific SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter. Analysis of two M. truncatula Tnt1 insertional mutants indicate that MtFE promotes flowering in LD and VLD and growth in all conditions tested. Expression of MtFTa1, MtFTb1, and MtFTb2 are reduced in Mtfe mutant (NF5076), correlating with its delayed flowering. The NF5076 mutant plants are much smaller than wild type indicating that MtFE is important for normal plant growth. The second mutant (NF18291) displays seedling lethality, like strong fe mutants. We searched for mutants in MtFTb1 and MtFTb2 identifying a Mtftb2 knock out Tnt1 mutant (NF20803). However, it did not flower significantly later than wild type. Previously, yeast-two-hybrid assays (Y2H) suggested that Arabidopsis FE interacted with CO and NUCLEAR FACTOR-Y (NF-Y)-like proteins to regulate FT. We found that MtFE interacts with CO and also M. truncatula NF-Y-like proteins in Y2H experiments. Our study indicates that despite the apparent absence of a functional MtCO-like gene, M. truncatula FE likely influences photoperiodic FT expression and flowering time in M. truncatula via a partially conserved mechanism with A. thaliana.