Role of sugars in the apical hook development of Arabidopsis etiolated seedlings.

KEY MESSAGE: The sugar supply in the medium affects the apical hook development of Arabidopsis etiolated seedlings. In addition, we provided the mechanism insights of this process. Dicotyledonous plants form an apical hook structure to shield their young cotyledons from mechanical damage as they emerge from the rough soil. Our findings indicate that sugar molecules, such as sucrose and glucose, are crucial for apical hook development. The presence of sucrose and glucose allows the apical hooks to be maintained for a longer period compared to those grown in sugar-free conditions, and this effect is dose-dependent. Key roles in apical hook development are played by several sugar metabolism pathways, including oxidative phosphorylation and glycolysis. RNA-seq data revealed an up-regulation of genes involved in starch and sucrose metabolism in plants grown in sugar-free conditions, while genes associated with phenylpropanoid metabolism were down-regulated. This study underscores the significant role of sugar metabolism in the apical hook development of etiolated Arabidopsis seedlings.

The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.

Light triggers an enhancement of global translation during photomorphogenesis in Arabidopsis, but little is known about the underlying mechanisms. The phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) at a conserved serine residue in the N-terminus has been shown as an important mechanism for the regulation of protein synthesis in mammalian and yeast cells. However, whether the phosphorylation of this residue in plant eIF2α plays a role in regulation of translation remains elusive. Here, we show that the quadruple mutant of SUPPRESSOR OF PHYA-105 family members (SPA1-SPA4) display repressed translation efficiency after light illumination. Moreover, SPA1 directly phosphorylates the eIF2α C-terminus under light conditions. The C-term-phosphorylated eIF2α promotes translation efficiency and photomorphogenesis, whereas the C-term-unphosphorylated eIF2α results in a decreased translation efficiency. We also demonstrate that the phosphorylated eIF2α enhances ternary complex assembly by promoting its affinity to eIF2ß and eIF2γ. This study reveals a unique mechanism by which light promotes translation via SPA1-mediated phosphorylation of the C-terminus of eIF2α in plants.

Mechanisms of calcium homeostasis orchestrate plant growth and immunity.

Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.

Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

A paternal signal induces endosperm proliferation upon fertilization in Arabidopsis.

In multicellular organisms, sexual reproduction relies on the formation of highly differentiated cells, the gametes, which await fertilization in a quiescent state. Upon fertilization, the cell cycle resumes. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that reinstate cell division in a fertilization-dependent manner are poorly understood in both animals and plants. Using Arabidopsis, we show that a sperm-derived signal induces the proliferation of a female gamete, the central cell, precisely upon fertilization. The central cell is arrested in S phase by the activity of the RETINOBLASTOMA RELATED1 (RBR1) protein. Upon fertilization, delivery of the core cell cycle component CYCD7;1 causes RBR1 degradation and thus S phase progression, ensuring the formation of functional endosperm and, consequently, viable seeds.

Time for growth.

Plants measure the duration of metabolic activity to promote rapid growth in long days.

Plants distinguish different photoperiods to independently control seasonal flowering and growth.

Plants measure daylength (photoperiod) to regulate seasonal growth and flowering. Photoperiodic flowering has been well studied, but less is known about photoperiodic growth. By using a mutant with defects in photoperiodic growth, we identified a seasonal growth regulation pathway that functions in long days in parallel to the canonical long-day photoperiod flowering mechanism. This is achieved by using distinct mechanisms to detect different photoperiods: The flowering pathway measures photoperiod as the duration of light intensity, whereas the growth pathway measures photoperiod as the duration of photosynthetic activity (photosynthetic period). Plants can then independently control expression of genes required for flowering or growth. This demonstrates that seasonal flowering and growth are dissociable, allowing them to be coordinated independently across seasons.

SHR and SCR coordinate root patterning and growth early in the cell cycle.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

The UBP5 histone H2A deubiquitinase counteracts PRCs-mediated repression to regulate Arabidopsis development.

Polycomb Repressive Complexes (PRCs) control gene expression through the incorporation of H2Aub and H3K27me3. In recent years, there is increasing evidence of the complexity of PRCs' interaction networks and the interplay of these interactors with PRCs in epigenome reshaping, which is fundamental to understand gene regulatory mechanisms. Here, we identified UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) as a chromatin player able to counteract the deposition of the two PRCs' epigenetic hallmarks in Arabidopsis thaliana. We demonstrated that UBP5 is a plant developmental regulator based on functional analyses of ubp5-CRISPR Cas9 mutant plants. UBP5 promotes H2A monoubiquitination erasure, leading to transcriptional de-repression. Furthermore, preferential association of UBP5 at PRC2 recruiting motifs and local H3K27me3 gaining in ubp5 mutant plants suggest the existence of functional interplays between UBP5 and PRC2 in regulating epigenome dynamics. In summary, acting as an antagonist of the pivotal epigenetic repressive marks H2Aub and H3K27me3, UBP5 provides novel insights to disentangle the complex regulation of PRCs' activities.

AtMYB50 regulates root cell elongation by upregulating PECTIN METHYLESTERASE INHIBITOR 8 in Arabidopsis thaliana.

Plant root development involves multiple signal transduction pathways. Notably, phytohormones like auxin and cytokinin are well characterized for their molecular mechanisms of action. Reactive oxygen species (ROS) serve as crucial signaling molecules in controlling root development. The transcription factor, UPBEAT1 (UPB1) is responsible for maintaining ROS homeostasis at the root tip, influencing the transition from cell proliferation to differentiation. While UPB1 directly regulates peroxidase expression to control ROS homeostasis, it targets genes other than peroxidases, suggesting its involvement in root growth through non-ROS signals. Our investigation focused on the transcription factor MYB50, a direct target of UPB1, in Arabidopsis thaliana. By analyzing multiple fluorescent proteins and conducting RNA-seq and ChIP-seq, we unraveled a step in the MYB50 regulatory gene network. This analysis, in conjunction with the UPB1 regulatory network, demonstrated that MYB50 directly regulates the expression of PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8). Overexpressing PMEI8, similar to the MYB50, resulted in reduced mature cell length. These findings establish MYB50 as a regulator of root growth within the UPB1 gene regulatory network. Our study presents a model involving transcriptional regulation by MYB50 in the UPB1 regulated root growth system and sheds light on cell elongation via pectin modification.

Air spaces bend light in plant stems.

Intercellular air spaces are necessary for phototropism in Arabidopsis thaliana.

Air channels create a directional light signal to regulate hypocotyl phototropism.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Cortical polarity ensures its own asymmetric inheritance in the stomatal lineage to pattern the leaf surface.

Asymmetric cell divisions specify differential cell fates across kingdoms. In metazoans, preferential inheritance of fate determinants into one daughter cell frequently depends on polarity-cytoskeleton interactions. Despite the prevalence of asymmetric divisions throughout plant development, evidence for analogous mechanisms that segregate fate determinants remains elusive. Here, we describe a mechanism in the Arabidopsis leaf epidermis that ensures unequal inheritance of a fate-enforcing polarity domain. By defining a cortical region depleted of stable microtubules, the polarity domain limits possible division orientations. Accordingly, uncoupling the polarity domain from microtubule organization during mitosis leads to aberrant division planes and accompanying cell identity defects. Our data highlight how a common biological module, coupling polarity to fate segregation through the cytoskeleton, can be reconfigured to accommodate unique features of plant development.

The WAK-like protein RFO1 acts as a sensor of the pectin methylation status in Arabidopsis cell walls to modulate root growth and defense.

Most organisms adjust their development according to the environmental conditions. For the majority, this implies the sensing of alterations to cell walls caused by different cues. Despite the relevance of this process, few molecular players involved in cell wall sensing are known and characterized. Here, we show that the wall-associated kinase-like protein RESISTANCE TO FUSARIUM OXYSPORUM 1 (RFO1) is required for plant growth and early defense against Fusarium oxysporum and functions by sensing changes in the pectin methylation levels in the cell wall. The RFO1 dwell time at the plasma membrane is affected by the pectin methylation status at the cell wall, regulating MITOGEN-ACTIVATED PROTEIN KINASE and gene expression. We show that the extracellular domain of RFO1 binds de-methylated pectin in vitro, whose distribution in the cell wall is altered during F. oxysporum infection. Further analyses also indicate that RFO1 is required for the BR-dependent plant growth alteration in response to inhibition of pectin de-methyl-esterase activity at the cell wall. Collectively, our work demonstrates that RFO1 is a sensor of the pectin methylation status that plays a unique dual role in plant growth and defense against vascular pathogens.

Export of defensive glucosinolates is key for their accumulation in seeds.

Plant membrane transporters controlling metabolite distribution contribute key agronomic traits1-6. To eliminate anti-nutritional factors in edible parts of crops, the mutation of importers can block the accumulation of these factors in sink tissues7. However, this often results in a substantially altered distribution pattern within the plant8-12, whereas engineering of exporters may prevent such changes in distribution. In brassicaceous oilseed crops, anti-nutritional glucosinolate defence compounds are translocated to the seeds. However, the molecular targets for export engineering of glucosinolates remain unclear. Here we identify and characterize members of the USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTER (UMAMIT) family-UMAMIT29, UMAMIT30 and UMAMIT31-in Arabidopsis thaliana as glucosinolate exporters with a uniport mechanism. Loss-of-function umamit29 umamit30 umamit31 triple mutants have a very low level of seed glucosinolates, demonstrating a key role for these transporters in translocating glucosinolates into seeds. We propose a model in which the UMAMIT uniporters facilitate glucosinolate efflux from biosynthetic cells along the electrochemical gradient into the apoplast, where the high-affinity H+-coupled glucosinolate importers GLUCOSINOLATE TRANSPORTERS (GTRs) load them into the phloem for translocation to the seeds. Our findings validate the theory that two differently energized transporter types are required for cellular nutrient homeostasis13. The UMAMIT exporters are new molecular targets to improve nutritional value of seeds of brassicaceous oilseed crops without altering the distribution of the defence compounds in the whole plant.

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root.

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.

RHO OF PLANT proteins are essential for pollen germination in Arabidopsis.

Pollen germination is a process of polarity establishment, through which a single and unique growth axis is established. Although most of the intracellular activities associated with pollen germination are controlled by RHO OF PLANTs (ROPs) and increased ROP activation accompanies pollen germination, a critical role of ROPs in this process has not yet been demonstrated. Here, by genomic editing of all 4 Arabidopsis (Arabidopsis thaliana) ROPs that are preferentially expressed in pollen, we showed that ROPs are essential for polarity establishment during pollen germination. We further identified and characterized 2 ROP effectors in pollen germination (REGs) through genome-wide interactor screening, boundary of ROP domain (BDR) members BDR8 and BDR9, whose functional loss also resulted in no pollen germination. BDR8 and BDR9 were distributed in the cytosol and the vegetative nucleus of mature pollen grains but redistributed to the plasma membrane (PM) of the germination site and to the apical PM of growing pollen tubes. We demonstrated that the PM redistribution of BDR8 and BDR9 during pollen germination relies on ROPs but not vice versa. Furthermore, enhanced expression of BDR8 partially restored germination of rop1 pollen but had no effects on that of the quadruple rop pollen, supporting their genetic epistasis. Results presented here demonstrate an ROP signaling route essential for pollen germination, which supports evolutionarily conserved roles of Rho GTPases in polarity establishment.

BBX30/miP1b and BBX31/miP1a form a positive feedback loop with ABI5 to regulate ABA-mediated postgermination seedling growth arrest.

In plants, the switch to autotrophic growth involves germination followed by postgermination seedling establishment. When environmental conditions are not favorable, the stress hormone abscisic acid (ABA) signals plants to postpone seedling establishment by inducing the expression of the transcription factor ABI5. The levels of ABI5 determine the efficiency of the ABA-mediated postgermination developmental growth arrest. The molecular mechanisms regulating the stability and activity of ABI5 during the transition to light are less known. Using genetic, molecular, and biochemical approach, we found that two B-box domain containing proteins BBX31 and BBX30 alongwith ABI5 inhibit postgermination seedling establishment in a partially interdependent manner. BBX31 and BBX30 are also characterized as microProteins miP1a and miP1b, respectively, based on their small size, single domain, and ability to interact with multidomain proteins. miP1a/BBX31 and miP1b/BBX30 physically interact with ABI5 to stabilize it and promote its binding to promoters of downstream genes. ABI5 reciprocally induces the expression of BBX30 and BBX31 by directly binding to their promoter. ABI5 and the two microProteins thereby form a positive feedback loop to promote ABA-mediated developmental arrest of seedlings.

Loss-of-Function Mutation of ACTIN-RELATED PROTEIN 6 (ARP6) Impairs Root Growth in Response to Salinity Stress.

H2A.Z-containing nucleosomes have been found to function in various developmental programs in Arabidopsis (e.g., floral transition, warm ambient temperature, and drought stress responses). The SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) complex is known to control the deposition of H2A.Z, and it has been unraveled that ACTIN-RELATED PROTEIN 6 (ARP6) is one component of this SWR1 complex. Previous studies showed that the arp6 mutant exhibited some distinguished phenotypes such as early flowering, leaf serration, elongated hypocotyl, and reduced seed germination rate in response to osmotic stress. In this study, we aimed to investigate the changes of arp6 mutant when the plants were grown in salt stress condition. The phenotypic observation showed that the arp6 mutant was more sensitive to salt stress than the wild type. Upon salt stress condition, this mutant exhibited attenuated root phenotypes such as shorter primary root length and fewer lateral root numbers. The transcript levels of stress-responsive genes, ABA INSENSITIVE 1 (ABI1) and ABI2, were found to be impaired in the arp6 mutant in comparison with wild-type plants in response to salt stress. In addition, a meta-analysis of published data indicated a number of genes involved in auxin response were induced in arp6 mutant grown in non-stress condition. These imply that the loss of H2A.Z balance (in arp6 mutant) may lead to change stress and auxin responses resulting in alternative root morphogenesis upon both normal and salinity stress conditions.