Mathematical Modeling of Photo- and Thermomorphogenesis in Plants.

Increased day lengths and warm conditions inversely affect plant growth by directly modulating nuclear phyB, ELF3, and COP1 levels. Quantitative measures of the hypocotyl length have been key to gaining a deeper understanding of this complex regulatory network, while similar quantitative data are the foundation for many studies in plant biology. Here, we explore the application of mathematical modeling, specifically ordinary differential equations (ODEs), to understand plant responses to these environmental cues. We provide a comprehensive guide to constructing, simulating, and fitting these models to data, using the law of mass action to study the evolution of molecular species. The fundamental principles of these models are introduced, highlighting their utility in deciphering complex plant physiological interactions and testing hypotheses. This brief introduction will not allow experimentalists without a mathematical background to run their own simulations overnight, but it will help them grasp modeling principles and communicate with more theory-inclined colleagues.

How plants protect themselves from ultraviolet-B radiation stress.

Ultraviolet-B (UV-B) radiation has a wavelength range of 280-315 nm. Plants perceive UV-B as an environmental signal and a potential abiotic stress factor that affects development and acclimation. UV-B regulates photomorphogenesis including hypocotyl elongation inhibition, cotyledon expansion, and flavonoid accumulation, but high intensity UV-B can also harm plants by damaging DNA, triggering accumulation of reactive oxygen species, and impairing photosynthesis. Plants have evolved "sunscreen" flavonoids that accumulate under UV-B stress to prevent or limit damage. The UV-B receptor UV RESISTANCE LOCUS 8 (UVR8) plays a critical role in promoting flavonoid biosynthesis to enhance UV-B stress tolerance. Recent studies have clarified several UVR8-mediated and UVR8-independent pathways that regulate UV-B stress tolerance. Here, we review these additions to our understanding of the molecular pathways involved in UV-B stress tolerance, highlighting the important roles of ELONGATED HYPOCOTYL 5, BRI1-EMS-SUPPRESSOR1, MYB DOMAIN PROTEIN 13, MAP KINASE PHOSPHATASE 1, and ATM- and RAD3-RELATED. We also summarize the known interactions with visible light receptors and the contribution of melatonin to UV-B stress responses. Finally, we update a working model of the UV-B stress tolerance pathway.

bHLH transcription factors LP1 and LP2 regulate longitudinal cell elongation.

Basic helix-loop-helix/helix-loop-helix (bHLH/HLH) transcription factors play substantial roles in plant cell elongation. In this study, two bHLH/HLH homologous proteins leaf related protein 1 and leaf-related protein 2 (AtLP1 and AtLP2) were identified in Arabidopsis thaliana. LP1 and LP2 play similar positive roles in longitudinal cell elongation. Both LP1 and LP2 overexpression plants exhibited long hypocotyls, elongated cotyledons, and particularly long leaf blades. The elongated leaves resulted from increased longitudinal cell elongation. lp1 and lp2 loss-of-function single mutants did not display distinct phenotypes, but the lp1lp2 double mutant showed decreased leaf length associated with less longitudinal polar cell elongation. Furthermore, the phenotype of lp1lp2 could be rescued by the expression of LP1 or LP2. Expression of genes related to cell elongation was upregulated in LP1 and LP2 overexpression plants but downregulated in lp1lp2 double mutant plants compared with that of wild type. LP1 and LP2 proteins could directly bind to the promoters of Longifolia1 (LNG1) and LNG2 to activate the expression of these cell elongation related genes. Both LP1 and LP2 could interact with two other bHLH/HLH proteins, IBH1 (ILI1 binding BHLH Protein1) and IBL1 (IBH1-like1), thereby suppressing the transcriptional activation of LP1 and LP2 to the target genes LNG1 and LNG2. Thus, our data suggested that LP1 and LP2 act as positive regulators to promote longitudinal cell elongation by activating the expression of LNG1 and LNG2 genes in Arabidopsis. Moreover, homodimerization of LP1 and LP2 may be essential for their function, and interaction between LP1/LP2 and other bHLH/HLH proteins may obstruct transcriptional regulation of target genes by LP1 and LP2.

Karrikins control seedling photomorphogenesis and anthocyanin biosynthesis through a HY5-BBX transcriptional module.

The butenolide molecule, karrikin (KAR), emerging in smoke of burned plant material, enhances light responses such as germination, inhibition of hypocotyl elongation, and anthocyanin accumulation in Arabidopsis. The KAR signaling pathway consists of KARRIKIN INSENSITIVE 2 (KAI2) and MORE AXILLARY GROWTH 2 (MAX2), which, upon activation, act in an SCF E3 ubiquitin ligase complex to target the downstream signaling components SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 2 (SMXL2) for degradation. How degradation of SMAX1 and SMXL2 is translated into growth responses remains unknown. Although light clearly influences the activity of KAR, the molecular connection between the two pathways is still poorly understood. Here, we demonstrate that the KAR signaling pathway promotes the activity of a transcriptional module consisting of ELONGATED HYPOCOTYL 5 (HY5), B-BOX DOMAIN PROTEIN 20 (BBX20), and BBX21. The bbx20 bbx21 mutant is largely insensitive to treatment with KAR2 , similar to a hy5 mutant, with regards to inhibition of hypocotyl elongation and anthocyanin accumulation. Detailed analysis of higher order mutants in combination with RNA-sequencing analysis revealed that anthocyanin accumulation downstream of SMAX1 and SMXL2 is fully dependent on the HY5-BBX module. However, the promotion of hypocotyl elongation by SMAX1 and SMXL2 is, in contrast to KAR2 treatment, only partially dependent on BBX20, BBX21, and HY5. Taken together, these results suggest that light- and KAR-dependent signaling intersect at the HY5-BBX transcriptional module.

Arabidopsis NF-YCs play dual roles in repressing brassinosteroid biosynthesis and signaling during light-regulated hypocotyl elongation.

Light functions as the primary environmental stimulus and brassinosteroids (BRs) as important endogenous growth regulators throughout the plant lifecycle. Photomorphogenesis involves a series of vital developmental processes that require the suppression of BR-mediated seedling growth, but the mechanism underlying the light-controlled regulation of the BR pathway remains unclear. Here, we reveal that nuclear factor YC proteins (NF-YCs) function as essential repressors of the BR pathway during light-controlled hypocotyl growth in Arabidopsis thaliana. In the light, NF-YCs inhibit BR biosynthesis by directly targeting the promoter of the BR biosynthesis gene BR6ox2 and repressing its transcription. NF-YCs also interact with BIN2, a critical repressor of BR signaling, and facilitate its stabilization by promoting its Tyr200 autophosphorylation, thus inhibiting the BR signaling pathway. Consistently, loss-of-function mutants of NF-YCs show etiolated growth and constitutive BR responses, even in the light. Our findings uncover a dual role of NF-YCs in repressing BR biosynthesis and signaling, providing mechanistic insights into how light antagonizes the BR pathway to ensure photomorphogenic growth in Arabidopsis.

Phytochrome-interacting factors interact with transcription factor CONSTANS to suppress flowering in rose.

As day-neutral (DN) woody perennial plants, the flowering time of roses (Rosa spp.) is assumed to be independent of the photoperiodic conditions; however, light responses of rose plants are not well understood. Chinese rose (Rosa chinensis) plants were grown under two light intensities (low light [LL], 92 µmol·m-2·s-1; or high light [HL], 278 µmol·m-2·s-1), and either with or without an end-of-day far-red (EOD-FR) treatment. Flowering was significantly delayed in the LL condition compared with the HL, but was not affected by EOD-FR treatment. The time until flowering positively corresponded with the mRNA and protein levels of phytochrome-interacting factors (PIFs; RcPIFs). The heterologous expression of RcPIF1, RcPIF3, or RcPIF4 in the Arabidopsis (Arabidopsis thaliana) pifq quadruple mutant partially rescued the mutant's shorter hypocotyl length. Simultaneous silencing of three RcPIFs in R. chinensis accelerated flowering under both LL and HL, with a more robust effect in LL, establishing RcPIFs as flowering suppressors in response to light intensity. The RcPIFs interacted with the transcription factor CONSTANS (RcCO) to form a RcPIFs-RcCO complex, which interfered with the binding of RcCO to the promoter of FLOWERING LOCUS T (RcFT), thereby inhibiting its expression. Furthermore, this inhibition was enhanced when RcPIFs were stabilized by LL, leading to delayed flowering under LL compared with HL. Our results not only revealed another layer of PIF functioning in the flowering of woody perennial plants, but also established a mechanism of light response in DN plants.

EIN3-Mediated Ethylene Signaling Attenuates Auxin Response during Hypocotyl Thermomorphogenesis.

The gaseous phytohormone ethylene plays vital roles in diverse developmental and environmental adaptation processes, such as fruit ripening, seedling establishment, mechanical stress tolerance and submergence escape. It is also known that in the light, ethylene promotes hypocotyl growth by stimulating the expression of PHYTOCHROME INTERACTING FACTOR3 (PIF3) transcription factor, which triggers microtubule reorganization during hypocotyl cell elongation. In particular, ethylene has been implicated in plant responses to warm temperatures in recent years. However, it is currently unclear how ethylene signals are functionally associated with hypocotyl thermomorphogenesis at the molecular level. Here, we show that ETHYLENE-INSENSITIVE3 (EIN3)-mediated ethylene signals attenuate hypocotyl thermomorphogenesis by suppressing auxin response. At warm temperatures, when the activity of the PIF4 thermomorphogenesis promoter is prominently high, the ethylene-activated EIN3 transcription factor directly induces the transcription of ARABIDOPSIS PP2C CLADE D7 (APD7) gene encoding a protein phosphatase that inactivates the plasma membrane (PM) H+-ATPase proton pumps. In conjunction with the promotive role of the PM H+-ATPases in hypocotyl cell elongation, our observations strongly support that the EIN3-directed induction of APD7 gene is linked with the suppression of auxin-induced cell expansion, leading to the reduction in thermomorphogenic hypocotyl growth. Our data demonstrate that APD7 acts as a molecular hub that integrates ethylene and auxin signals into hypocotyl thermomorphogenesis. We propose that the ethylene-auxin signaling crosstalks via the EIN3-APD7 module facilitate the fine-tuning of hypocotyl thermomorphogenesis under natural environments, which often fluctuate in a complex manner.

Brassinosteroids Influence Arabidopsis Hypocotyl Graviresponses through Changes in Mannans and Cellulose.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base.

An auxin-mediated regulatory framework for wound-induced adventitious root formation in tomato shoot explants.

Adventitious roots (ARs) are produced from non-root tissues in response to different environmental signals, such as abiotic stresses, or after wounding, in a complex developmental process that requires hormonal crosstalk. Here, we characterized AR formation in young seedlings of Solanum lycopersicum cv. 'Micro-Tom' after whole root excision by means of physiological, genetic and molecular approaches. We found that a regulated basipetal auxin transport from the shoot and local auxin biosynthesis triggered by wounding are both required for the re-establishment of internal auxin gradients within the vasculature. This promotes cell proliferation at the distal cambium near the wound in well-defined positions of the basal hypocotyl and during a narrow developmental window. In addition, a pre-established pattern of differential auxin responses along the apical-basal axis of the hypocotyl and an as of yet unknown cell-autonomous inhibitory pathway contribute to the temporal and spatial patterning of the newly formed ARs on isolated hypocotyl explants. Our work provides an experimental outline for the dissection of wound-induced AR formation in tomato, a species that is suitable for molecular identification of gene regulatory networks via forward and reverse genetics approaches.

Plant grafting relieves asymmetry of jasmonic acid response induced by wounding between scion and rootstock in tomato hypocotyl.

Plant grafting is a sequential wound healing process. However, whether wounding induces a different jasmonic acid (JA) response within half a day (12 h) after grafting or non-grafting remains unclear. Using the tomato hypocotyl grafting method, we show that grafting alleviates the asymmetrical accumulation of JA and jasmonic acid isoleucine conjugate (JA-Ile) in scion and rootstock caused by wounding, and from 2 h after tomato micrografting, grafting obviously restored the level of JA-Ile in the scion and rootstock. Meanwhile, five JA-related genes, SlLOX11, SlAOS, SlCOI1, SlLAPA and SlJA2L, are detected and show significant changes in transcriptional expression patterns within 12 h of grafting, from asymmetrical to symmetrical, when the expression of 30 JA- and defense-related genes were analyzed. The results indicated that grafting alleviates the asymmetrical JA and defense response between scion and rootstock of the tomato hypocotyl within 12 h as induced by wounding. Moreover, we demonstrate that in the very early hours after grafting, JA-related genes may be involved in a molecular mechanism that changes asymmetrical expression as induced by wounding between scion and rootstock, thereby promoting wound healing and grafting success.

Cell Wall Composition as a Marker of the Reprogramming of the Cell Fate on the Example of a Daucus carota (L.) Hypocotyl in Which Somatic Embryogenesis Was Induced.

Changes in the composition of the cell walls are postulated to accompany changes in the cell's fate. We check whether there is a relationship between the presence of selected pectic, arabinogalactan proteins (AGPs), and extensins epitopes and changes in cell reprogramming in order to answer the question of whether they can be markers accompanying changes of cell fate. Selected antibodies were used for spatio-temporal immunolocalization of wall components during the induction of somatic embryogenesis. Based on the obtained results, it can be concluded that (1) the LM6 (pectic), LM2 (AGPs) epitopes are positive markers, but the LM5, LM19 (pectic), JIM8, JIM13 (AGPs) epitopes are negative markers of cells reprogramming to the meristematic/pluripotent state; (2) the LM8 (pectic), JIM8, JIM13, LM2 (AGPs) and JIM11 (extensin) epitopes are positive markers, but LM6 (pectic) epitope is negative marker of cells undergoing detachment; (3) JIM4 (AGPs) is a positive marker, but LM5 (pectic), JIM8, JIM13, LM2 (AGPs) are negative markers for pericycle cells on the xylem pole; (4) LM19, LM20 (pectic), JIM13, LM2 (AGPs) are constitutive wall components, but LM6, LM8 (pectic), JIM4, JIM8, JIM16 (AGPs), JIM11, JIM12 and JIM20 (extensins) are not constitutive wall components; (5) the extensins do not contribute to the cell reprogramming.

Salt-tolerant and -sensitive seedlings exhibit noteworthy differences in lipolytic events in response to salt stress.

Present findings hypothesize that salt-tolerant and -sensitive oilseed plants are expected to exhibit deviant patterns of growth through lipolytic events in seedling cotyledons. It reports the growth response and different lipolytic mechanisms operating during oil body (OB) mobilization in the seedling cotyledons of salt-tolerant (DRSH 1) and salt-sensitive (PSH 1962) varieties of sunflower (Helianthus annuus L.). Salt tolerance or sensitivity to 120 mM NaCl correlates with high proteolytic degradation of OB membrane proteins, particularly oleosins, whereas salt-sensitive seedling cotyledons exhibit negligible proteolytic activity, thereby retaining OB membrane integrity for a longer time. High lipoxygenase (LOX) activity and its further upregulation by salt stress are the unique features of salt-sensitive sunflower seedlings. Salt-tolerant seedling cotyledons exhibit noteworthy modulation of phospholipase-D (PLD) activity by salt stress. Salt-sensitive seedling cotyledons exhibit higher lipase activity than salt-sensitive ones and enzyme activity is downregulated by salt stress. Salt-sensitive variety exhibits higher lipid accumulation and faster lipid mobilization with seedling development than salt-tolerant variety. Accumulation of oleic and linoleic acid in the seedling cotyledons of salt-tolerant and sensitive varieties exhibits differential sensitivity to salt stress. Novel detection of hexanoic acid (6:0) is a noteworthy feature as a response to salt stress in salt-sensitive variety. These findings, thus, provide new information on long-distance salt stress sensing mechanisms at seedling stage of plant development.

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

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

Shade avoidance responses become more aggressive in warm environments.

When exposed to neighbour cues, competitive plants increase stem growth to reduce the degree of current or future shade. The aim of this work is to investigate the impact of weather conditions on the magnitude of shade avoidance responses in Arabidopsis thaliana. We first generated a growth rate database under controlled conditions and elaborated a model that predicts daytime hypocotyl growth as a function of the activity of the main photosensory receptors (phytochromes A and B, cryptochromes 1 and 2) in combination with light and temperature inputs. We then incorporated the action of thermal amplitude to account for its effect on selected genotypes, which correlates with the dynamics of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR 4. The model predicted growth rate in the field with reasonable accuracy. Thus, we used the model in combination with a worldwide data set of current and future whether conditions. The analysis predicted enhanced shade avoidance responses as a result of higher temperatures due to the geographical location or global warming. Irradiance and thermal amplitude had no effects. These trends were also observed for our local growth rate measurements. We conclude that, if water and nutrients do not become limiting, warm environments enhance the shade avoidance response.

Cryptochrome-mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis.

Both phototropins (phot1 and phot2) and cryptochromes (cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids (GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high-intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3 (nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose-containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3. Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.

Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA.

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development are completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes.

The ABA-induced soybean ERF transcription factor gene GmERF75 plays a role in enhancing osmotic stress tolerance in Arabidopsis and soybean.

BACKGROUND: Ethylene-responsive factors (ERFs) play important roles in plant growth and development and the response to adverse environmental factors, including abiotic and biotic stresses. RESULTS: In the present study, we identified 160 soybean ERF genes distributed across 20 chromosomes that could be clustered into eight groups based on phylogenetic relationships. A highly ABA-responsive ERF gene, GmERF75, belonging to Group VII was further characterized. Subcellular localization analysis showed that the GmERF75 protein is localized in the nucleus, and qRT-PCR results showed that GmERF75 is responsive to multiple abiotic stresses and exogenous hormones. GmERF75-overexpressing Arabidopsis lines showed higher chlorophyll content compared to WT and mutants under osmotic stress. Two independent Arabidopsis mutations of AtERF71, a gene homologous to GmERF75, displayed shorter hypocotyls, and overexpression of GmERF75 in these mutants could rescue the short hypocotyl phenotypes. Overexpressing GmERF75 in soybean hairy roots improved root growth under exogenous ABA and salt stress. CONCLUSIONS: These results suggested that GmERF75 is an important plant transcription factor that plays a critical role in enhancing osmotic tolerance in both Arabidopsis and soybean.

Nonsense-Mediated mRNA Decay Deficiency Affects the Auxin Response and Shoot Regeneration in Arabidopsis.

Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.