High-Throughput Spike Detection in Greenhouse Cultivated Grain Crops with Attention Mechanisms-Based Deep Learning Models.

Detection of spikes is the first important step toward image-based quantitative assessment of crop yield. However, spikes of grain plants occupy only a tiny fraction of the image area and often emerge in the middle of the mass of plant leaves that exhibit similar colors to spike regions. Consequently, accurate detection of grain spikes renders, in general, a non-trivial task even for advanced, state-of-the-art deep neural networks (DNNs). To improve pattern detection in spikes, we propose architectural changes to Faster-RCNN (FRCNN) by reducing feature extraction layers and introducing a global attention module. The performance of our extended FRCNN-A vs. conventional FRCNN was compared on images of different European wheat cultivars, including "difficult" bushy phenotypes from 2 different phenotyping facilities and optical setups. Our experimental results show that introduced architectural adaptations in FRCNN-A helped to improve spike detection accuracy in inner regions. The mean average precision (mAP) of FRCNN and FRCNN-A on inner spikes is 76.0% and 81.0%, respectively, while on the state-of-the-art detection DNNs, Swin Transformer mAP is 83.0%. As a lightweight network, FRCNN-A is faster than FRCNN and Swin Transformer on both baseline and augmented training datasets. On the FastGAN augmented dataset, FRCNN achieved a mAP of 84.24%, FRCNN-A attained a mAP of 85.0%, and the Swin Transformer achieved a mAP of 89.45%. The increase in mAP of DNNs on the augmented datasets is proportional to the amount of the IPK original and augmented images. Overall, this study indicates a superior performance of attention mechanisms-based deep learning models in detecting small and subtle features of grain spikes.

Cytokinins - regulators of de novo shoot organogenesis.

Plants, unlike animals, possess a unique developmental plasticity, that allows them to adapt to changing environmental conditions. A fundamental aspect of this plasticity is their ability to undergo postembryonic de novo organogenesis. This requires the presence of regulators that trigger and mediate specific spatiotemporal changes in developmental programs. The phytohormone cytokinin has been known as a principal regulator of plant development for more than six decades. In de novo shoot organogenesis and in vitro shoot regeneration, cytokinins are the prime candidates for the signal that determines shoot identity. Both processes of de novo shoot apical meristem development are accompanied by changes in gene expression, cell fate reprogramming, and the switching-on of the shoot-specific homeodomain regulator, WUSCHEL. Current understanding about the role of cytokinins in the shoot regeneration will be discussed.

RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

bHLH heterodimer complex variations regulate cell proliferation activity in the meristems of Arabidopsis thaliana.

Root, shoot, and lateral meristems are the main regions of cell proliferation in plants. It has been proposed that meristems might have evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that basic helix-loop-helix (bHLH) transcription factor heterodimers formed by members of the TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades are general regulators of cell proliferation in all meristems. Yet, genetics and expression analyses suggest specific functions of these transcription factors in distinct meristems, possibly due to their expression domains determining heterodimer complex variations within meristems, and to a certain extent to the absence of some of them in a given meristem. Target gene specificity analysis for heterodimer complexes focusing on the LONELY GUY gene targets further suggests differences in transcriptional responses through heterodimer diversification that could allow a common bHLH heterodimer complex module to contribute to cell proliferation control in multiple meristems.

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

Aluminum Stress Induces Irreversible Proteomic Changes in the Roots of the Sensitive but Not the Tolerant Genotype of Triticale Seedlings.

Triticale is a wheat-rye hybrid with a higher abiotic stress tolerance than wheat and is better adapted for cultivation in light-type soils, where aluminum ions are present as Al-complexes that are harmful to plants. The roots are the first plant organs to contact these ions and the inhibition of root growth is one of the first plant reactions. The proteomes of the root apices in Al-tolerant and -sensitive plants were investigated to compare their regeneration effects following stress. The materials used in this study consisted of seedlings of three triticale lines differing in Al3+ tolerance, first subjected to aluminum ion stress and then recovered. Two-dimensional electrophoresis (2-DE) was used for seedling root protein separation followed by differential spot analysis using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS/MS). The plants' tolerance to the stress was evaluated based on biometric screening of seedling root regrowth upon regeneration. Our results suggest that the Al-tolerant genotype can recover, without differentiation of proteome profiles, after stress relief, contrary to Al-sensitive genotypes that maintain the proteome modifications caused by unfavorable environments.

Histidine kinase inhibitors impair shoot regeneration in Arabidopsis thaliana via cytokinin signaling and SAM patterning determinants.

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754.

Auxin Does the SAMba: Auxin Signaling in the Shoot Apical Meristem.

Plants, in contrast to animals, are unique in their capacity to postembryonically develop new organs due to the activity of stem cell populations, located in specialized tissues called meristems. Above ground, the shoot apical meristem generates aerial organs and tissues throughout plant life. It is well established that auxin plays a central role in the functioning of the shoot apical meristem. Auxin distribution in the meristem is not uniform and depends on the interplay between biosynthesis, transport, and degradation. Auxin maxima and minima are created, and result in transcriptional outputs that drive the development of new organs and contribute to meristem maintenance. To uncover and understand complex signaling networks such as the one regulating auxin responses in the shoot apical meristem remains a challenge. Here, we will discuss our current understanding and point to important research directions for the future.

Developmental roles of Auxin Binding Protein 1 in Arabidopsis thaliana.

Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.

Cell-surface receptors enable perception of extracellular cytokinins.

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

Cytokinin signalling regulates organ identity via the AHK4 receptor in Arabidopsis.

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.

Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis.

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

What Has Been Seen Cannot Be Unseen-Detecting Auxin In Vivo.

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

Cytokinins influence root gravitropism via differential regulation of auxin transporter expression and localization in Arabidopsis.

Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism.

Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development.

Integrating important environmental signals with intrinsic developmental programmes is a crucial adaptive requirement for plant growth, survival, and reproduction. Key environmental cues include changes in several light variables, while important intrinsic (and highly interactive) regulators of many developmental processes include the phytohormones cytokinins (CKs) and ethylene. Here, we discuss the latest discoveries regarding the molecular mechanisms mediating CK/ethylene crosstalk at diverse levels of biosynthetic and metabolic pathways and their complex interactions with light. Furthermore, we summarize evidence indicating that multiple hormonal and light signals are integrated in the multistep phosphorelay (MSP) pathway, a backbone signalling pathway in plants. Inter alia, there are strong overlaps in subcellular localizations and functional similarities in components of these pathways, including receptors and various downstream agents. We highlight recent research demonstrating the importance of CK/ethylene/light crosstalk in selected aspects of plant development, particularly seed germination and early seedling development. The findings clearly demonstrate the crucial integration of plant responses to phytohormones and adaptive responses to environmental cues. Finally, we tentatively identify key future challenges to refine our understanding of the molecular mechanisms mediating crosstalk between light and hormonal signals, and their integration during plant life cycles.

Cytokinin and auxin interactions in plant development: metabolism, signalling, transport and gene expression.

Auxin and cytokinins have been identified as key regulators of plant development. Recently, these phytohormones have been shown to interact during important developmental processes, including positioning, identity acquisition and maintenance of meristem organizing centres, regulation of balance between cell division and differentiation, and postembryonic de novo organogenesis. Here, we discuss recent advances in our understanding of the underlying molecular mechanisms at the levels of regulating metabolism, signalling, gene expression and protein stability.

Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux.

Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta. Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.