NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.

Genomic basis of the distinct biosynthesis of ß-glucogallin, a biochemical marker for hydrolyzable tannin production, in three oak species.

Hydrolyzable tannins (HTs), predominant polyphenols in oaks, are widely used in grape wine aging, feed additives, and human healthcare. However, the limited availability of a high-quality reference genome of oaks greatly hampered the recognition of the mechanism of HT biosynthesis. Here, high-quality reference genomes of three Asian oak species (Quercus variabilis, Quercus aliena, and Quercus dentata) that have different HT contents were generated. Multi-omics studies were carried out to identify key genes regulating HT biosynthesis. In vitro enzyme activity assay was also conducted. Dual-luciferase and yeast one-hybrid assays were used to reveal the transcriptional regulation. Our results revealed that ß-glucogallin was a biochemical marker for HT production in the cupules of the three Asian oaks. UGT84A13 was confirmed as the key enzyme for ß-glucogallin biosynthesis. The differential expression of UGT84A13, rather than enzyme activity, was the main reason for different ß-glucogallin and HT accumulation. Notably, sequence variations in UGT84A13 promoters led to different trans-activating activities of WRKY32/59, explaining the different expression patterns of UGT84A13 among the three species. Our findings provide three high-quality new reference genomes for oak trees and give new insights into different transcriptional regulation for understanding ß-glucogallin and HT biosynthesis in closely related oak species.

The plant trans-Golgi network component ECHIDNA regulates defense, cell death, and endoplasmic reticulum stress.

The trans-Golgi network (TGN) acts as a central platform for sorting and secreting various cargoes to the cell surface, thus being essential for the full execution of plant immunity. However, the fine-tuned regulation of TGN components in plant defense and stress response has been not fully elucidated. Our study revealed that despite largely compromising penetration resistance, the loss-of-function mutation of the TGN component protein ECHIDNA (ECH) induced enhanced postinvasion resistance to powdery mildew in Arabidopsis thaliana. Genetic and transcriptome analyses and hormone profiling demonstrated that ECH loss resulted in salicylic acid (SA) hyperaccumulation via the ISOCHORISMATE SYNTHASE 1 biosynthesis pathway, thereby constitutively activating SA-dependent innate immunity that was largely responsible for the enhanced postinvasion resistance. Furthermore, the ech mutant displayed accelerated SA-independent spontaneous cell death and constitutive POWDERY MILDEW RESISTANCE 4-mediated callose depositions. In addition, ECH loss led to a chronically prolonged endoplasmic reticulum stress in the ech mutant. These results provide insights into understanding the role of TGN components in the regulation of plant immunity and stress responses.

Molecular Advances of Bud Dormancy in Trees.

Seasonal bud dormancy in perennial woody plants is a crucial and intricate process that is vital for the survival and development of plants. Over the past few decades, significant advancements have been made in understanding many features of bud dormancy, particularly in model species, where certain molecular mechanisms underlying this process have been elucidated. In this review, we provide an overview of recent molecular progress in understanding bud dormancy in trees, with a specific emphasis on the integration of common signaling and molecular mechanisms identified across different tree species. Additionally, we address some challenges that have emerged in the in-depth understanding of bud dormancy and offer insights for future studies.

The RALF1-FERONIA interaction modulates endocytosis to mediate control of root growth in Arabidopsis.

The interaction between the receptor-like kinase (RLK) FERONIA (FER) and the secreted peptide RAPID ALKALINIZATION FACTOR1 (RALF1) is vital for development and stress responses in Arabidopsis Ligand-induced membrane dynamics affect the function of several RLKs, but the effects of the RALF1-FER interaction on the dynamics of FER and the ensuing effects on its functionality are poorly understood. Here, we show that RALF1 modulated the dynamics and partitioning of FER-GFP at the plasma membrane (PM). Moreover, FER was internalized by both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) under steady-state conditions. After RALF1 treatment, FER-GFP internalization was primarily enhanced via the CME pathway, raising FER-GFP levels in the vacuole. RALF1 treatment also modulated trafficking of other PM proteins, such as PIN2-GFP and BRI1-GFP, increasing their vacuolar levels by enhancing their internalization. Importantly, blocking CME attenuated RALF1-mediated root growth inhibition independently of RALF1-induced early signaling, suggesting that the RALF1 can also exert its effects via the CME pathway. These findings reveal that the RALF1-FER interaction modulates plant growth and development, and this might also involve endocytosis of PM proteins.

A genetic framework for regulation and seasonal adaptation of shoot architecture in hybrid aspen.

Shoot architecture is critical for optimizing plant adaptation and productivity. In contrast with annuals, branching in perennials native to temperate and boreal regions must be coordinated with seasonal growth cycles. How branching is coordinated with seasonal growth is poorly understood. We identified key components of the genetic network that controls branching and its regulation by seasonal cues in the model tree hybrid aspen. Our results demonstrate that branching and its control by seasonal cues is mediated by mutually antagonistic action of aspen orthologs of the flowering regulators TERMINAL FLOWER 1 (TFL1) and APETALA1 (LIKE APETALA 1/LAP1). LAP1 promotes branching through local action in axillary buds. LAP1 acts in a cytokinin-dependent manner, stimulating expression of the cell-cycle regulator AIL1 and suppressing BRANCHED1 expression to promote branching. Short photoperiod and low temperature, the major seasonal cues heralding winter, suppress branching by simultaneous activation of TFL1 and repression of the LAP1 pathway. Our results thus reveal the genetic network mediating control of branching and its regulation by environmental cues facilitating integration of branching with seasonal growth control in perennial trees.

Rho-of-plant activated root hair formation requires Arabidopsis YIP4a/b gene function.

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

Long-range mobile signals mediate seasonal control of shoot growth.

In perennial plants, seasonal shifts provide cues that control adaptive growth patterns of the shoot apex. However, where these seasonal cues are sensed and communicated to the shoot apex remains unknown. We demonstrate that systemic signals from leaves play key roles in seasonal control of shoot growth in model tree hybrid aspen. Grafting experiments reveal that the tree ortholog of Arabidopsis flowering time regulator FLOWERING LOCUS T (FT) and the plant hormone gibberellic acid (GA) systemically convey seasonal cues to the shoot apex. GA (unlike FT) also acts locally in shoot apex, downstream of FT in seasonal growth control. At the shoot apex, antagonistic factors-LAP1, a target of FT and the FT antagonist TERMINAL FLOWER 1 (TFL1)-act locally to promote and suppress seasonal growth, respectively. These data reveal seasonal changes perceived in leaves that are communicated to the shoot apex by systemic signals that, in concert with locally acting components, control adaptive growth patterns.

Katanin-Dependent Microtubule Ordering in Association with ABA Is Important for Root Hydrotropism.

Root hydrotropism refers to root directional growth toward soil moisture. Cortical microtubule arrays are essential for determining the growth axis of the elongating cells in plants. However, the role of microtubule reorganization in root hydrotropism remains elusive. Here, we demonstrate that the well-ordered microtubule arrays and the microtubule-severing protein KATANIN (KTN) play important roles in regulating root hydrotropism in Arabidopsis. We found that the root hydrotropic bending of the ktn1 mutant was severely attenuated but not root gravitropism. After hydrostimulation, cortical microtubule arrays in cells of the elongation zone of wild-type (WT) Col-0 roots were reoriented from transverse into an oblique array along the axis of cell elongation, whereas the microtubule arrays in the ktn1 mutant remained in disorder. Moreover, we revealed that abscisic acid (ABA) signaling enhanced the root hydrotropism of WT and partially rescued the oryzalin (a microtubule destabilizer) alterative root hydrotropism of WT but not ktn1 mutants. These results suggest that katanin-dependent microtubule ordering is required for root hydrotropism, which might work downstream of ABA signaling pathways for plant roots to search for water.

Independent yet overlapping pathways ensure the robustness and responsiveness of trans-Golgi network functions in Arabidopsis.

The trans-Golgi-network (TGN) has essential housekeeping functions in secretion, endocytosis and protein sorting, but also more specialized functions in plant development. How the robustness of basal TGN function is ensured while specialized functions are differentially regulated is poorly understood. Here, we investigate two key regulators of TGN structure and function, ECHIDNA and the Transport Protein Particle II (TRAPPII) tethering complex. An analysis of physical, network and genetic interactions suggests that two network communities are implicated in TGN function and that ECHIDNA and TRAPPII belong to distinct yet overlapping pathways. Whereas ECHIDNA and TRAPPII colocalized at the TGN in interphase cells, their localization diverged in dividing cells. Moreover, ECHIDNA and TRAPPII localization patterns were mutually independent. TGN structure, endocytosis and sorting decisions were differentially impacted in echidna and trappii mutants. Our analyses point to a partitioning of specialized TGN functions, with ECHIDNA being required for cell elongation and TRAPPII for cytokinesis. Two independent pathways able to compensate for each other might contribute to the robustness of TGN housekeeping functions and to the responsiveness and fine tuning of its specialized functions.

A hundred years after: endodormancy and the chilling requirement in subtropical trees.

Endodormancy and the related chilling requirement synchronize the seasonal development of trees from the boreal and temperate regions under the climatic conditions prevailing at their native growing sites. The phenomenon of endodormancy has been known at the whole-plant level for 100 years, and in the last couple of decades, insights into the physiological and molecular basis of endodormancy and its release have also been obtained. Intriguingly, recent studies have shown experimentally that subtropical trees also show endodormancy and a chilling requirement. Motivated by the climatic differences between the subtropical and more northern zones, here we address the similarities and differences in endodormancy between trees growing in the subtropical zone and those growing in more northern zones.

The chromatin-modifying protein HUB2 is involved in the regulation of lignin composition in xylem vessels.

PIRIN2 (PRN2) was earlier reported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis thaliana. In the present study, we report yeast two-hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by ß-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis-GC/MS. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier transform infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analysis of the hub2 prn2 double mutant, support the antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic gene FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localized in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin-promoting function of HUB2 is antagonized by PRN2 for the benefit of the guaiacyl (G)-type lignin enrichment of the neighbouring xylem vessel elements.

Ethylene Regulates Differential Growth via BIG ARF-GEF-Dependent Post-Golgi Secretory Trafficking in Arabidopsis.

During early seedling development, the shoot apical meristem is protected from damage as the seedling emerges from soil by the formation of apical hook. Hook formation requires differential growth across the epidermis below the meristem in the hypocotyl. The plant hormones ethylene and auxin play key roles during apical hook development by controlling differential growth. We provide genetic and cell biological evidence for the role of ADP-ribosylation factor 1 (ARF1)-GTPase and its effector ARF-guanine-exchange factors (GEFs) of the Brefeldin A-inhibited GEF (BIG) family and GNOM in ethylene- and auxin-mediated control of hook development. We show that ARF-GEF GNOM acts early, whereas BIG ARF-GEFs act at a later stage of apical hook development. We show that the localization of ARF1 and BIG4 at the trans-Golgi network (TGN) depends on ECHIDNA (ECH), a plant homolog of yeast Triacylglycerol lipase (TLG2/SYP4) interacting protein Tgl2-Vesicle Protein 23 (TVP23). BIGs together with ECH and ARF1 mediate the secretion of AUX1 influx carrier to the plasma membrane from the TGN during hook development and defects in BIG or ARF1 result in insensitivity to ethylene. Thus, our data indicate a division of labor within the ARF-GEF family in mediating differential growth with GNOM acting during the formation phase whereas BIGs act during the hook maintenance phase downstream of plant hormone ethylene.

Abscisic acid signalling mediates biomass trade-off and allocation in poplar.

Abscisic acid (ABA) is a well known stress hormone regulating drought adaptation of plants. Here, we hypothesised that genetic engineering of genes involved in ABA stress signalling and photoperiodic regulation affected drought resistance by trade-off with biomass production in perennial poplar trees. We grew Populus tremula × tremuloides wild-type (T89) and various transgenic lines (two transformation events of 35S::abi1-1, 35S::RCAR, RCAR:RNAi, 35S::ABI3, 35S::AREB3, 35S::FDL1, FDL1:RNAi, 35S::FDL2 and FDL2:RNAi) outdoors and exposed them to drought in the second growth period. After the winter, the surviving lines showed a huge variation in stomatal conductance, leaf size, whole-plant leaf area, tree height, stem diameter, and biomass. Whole-plant leaf area was a strong predictor for woody biomass production. The 35S::AREB3 lines were compromised in biomass production under well irrigated conditions compared with wild-type poplars but were resilient to drought. ABA signalling regulated FDL1 and FDL2 expression under stress. Poplar lines overexpressing FDL1 or FDL2 were drought-sensitive; they shed leaves and lost root biomass, whereas the FDL RNAi lines showed higher biomass allocation to roots under drought. These results assign a new function in drought acclimation to FDL genes aside from photoperiodic regulation. Our results imply a critical role for ABA-mediated processes in balancing biomass production and climate adaptation.

Storage lipid accumulation is controlled by photoperiodic signal acting via regulators of growth cessation and dormancy in hybrid aspen.

The signalling pathways that control seasonal modulation of carbon metabolism in perennial plants are poorly understood. Using genetic, metabolic and natural variation approaches, we identify factors mediating photoperiodic control of storage lipid accumulation in the model tree hybrid aspen (Populus tremula × tremuloides). We characterized lipid accumulation in transgenic hybrid aspen with impaired photoperiodic and hormonal responses. Genome-wide association mapping was performed in Swedish aspen (P. tremula) genotypes to determine genetic loci associated with genotype variation in lipid content. Our data show that the storage lipid triacylglycerol (TAG) accumulates in cambial meristem and pith rays of aspen in response to photoperiodic signal controlling growth cessation and dormancy induction. We show that photoperiodic control of TAG accumulation is mediated by the FLOWERING LOCUS T/CONSTANS module, which also controls the induction of growth cessation. Hormonal and chromatin remodelling pathways also contribute to TAG accumulation by photoperiodic signal. Natural variation exists in lipid accumulation that is controlled by input from multiple loci. Our data shed light on how the control of storage metabolism is temporally coordinated with growth cessation and dormancy by photoperiodic signal, and reveals that storage lipid accumulation between seeds and perennating organs of trees may involve distinct regulatory circuits.

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha.

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.

Dual role of tree florigen activation complex component FD in photoperiodic growth control and adaptive response pathways.

A complex consisting of evolutionarily conserved FD, flowering locus T (FT) proteins is a regulator of floral transition. Intriguingly, FT orthologs are also implicated in developmental transitions distinct from flowering, such as photoperiodic control of bulbing in onions, potato tuberization, and growth cessation in trees. However, whether an FT-FD complex participates in these transitions and, if so, its mode of action, are unknown. We identified two closely related FD homologs, FD-like 1 (FDL1) and FD-like 2 (FDL2), in the model tree hybrid aspen. Using gain of function and RNAi-suppressed FDL1 and FDL2 transgenic plants, we show that FDL1 and FDL2 have distinct functions and a complex consisting of FT and FDL1 mediates in photoperiodic control of seasonal growth. The downstream target of the FT-FD complex in photoperiodic control of growth is Like AP1 (LAP1), a tree ortholog of the floral meristem identity gene APETALA1. Intriguingly, FDL1 also participates in the transcriptional control of adaptive response and bud maturation pathways, independent of its interaction with FT, presumably via interaction with abscisic acid insensitive 3 (ABI3) transcription factor, a component of abscisic acid (ABA) signaling. Our data reveal that in contrast to its primary role in flowering, FD has dual roles in the photoperiodic control of seasonal growth and stress tolerance in trees. Thus, the functions of FT and FD have diversified during evolution, and FD homologs have acquired roles that are independent of their interaction with FT.

Genome Wide Binding Site Analysis Reveals Transcriptional Coactivation of Cytokinin-Responsive Genes by DELLA Proteins.

The ability of plants to provide a plastic response to environmental cues relies on the connectivity between signaling pathways. DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis.

Photoperiod- and temperature-mediated control of phenology in trees - a molecular perspective.

Contents 511 I. 511 II. 512 III. 513 IV. 513 V. 517 VI. 517 VII. 521 VIII. 521 Acknowledgements 521 References 521 SUMMARY: Trees growing in boreal and temperate regions synchronize their growth with seasonal climatic changes in adaptive responses that are essential for their survival. These trees cease growth before the winter and establish a dormant state during which growth cessation is maintained by repression of responses to growth-promotive signals. Reactivation of growth in the spring follows the release from dormancy promoted by prolonged exposure to low temperature during the winter. The timing of the key events and regulation of the molecular programs associated with the key stages of the annual growth cycle are controlled by two main environmental cues: photoperiod and temperature. Recently, key components mediating photoperiodic control of growth cessation and bud set have been identified, and striking similarities have been observed in signaling pathways controlling growth cessation in trees and floral transition in Arabidopsis. Although less well understood, the regulation of bud dormancy and bud burst may involve cell-cell communication and chromatin remodeling. Here, we discuss current knowledge of the molecular-level regulation of the annual growth cycle of woody trees in temperate and boreal regions, and identify key questions that need to be addressed in the future.