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In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

Secondary root emergence is a crucial trait that shapes the plant’s underground system. Virtually every developmental step of root primordium morphogenesis is controlled by auxin. However, how the hormone controls cell separation in primordium-overlaying tissues through wall loosening is poorly understood. Here, we took advantage of white lupin and its spectacular cluster root development to assess the contribution of auxin to this process. We show that auxin’s positive role on rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes. Downregulation of a pectinolytic enzyme gene expressed in cells surrounding the primordium resulted in delayed emergence. Pectins were demethylesterified in the emergence zone and auxin treatment further enhanced this effect. Additionally, we report specific rhamnogalacturonan-I modifications during cortical cell separation. In conclusion, we propose a model in which auxin has a dual role during rootlet emergence: Firstly, through active pectin demethylesterification and secondly by regulating the expression of cell wall remodeling enzymes.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula., Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.

In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.

Summary Single‐cell RNA‐seq (scRNA‐seq) has been highlighted as a powerful tool for the description of human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single cells were divided into eight cell clusters based on reported marker genes by applying scRNA‐seq. Further, a pseudo‐time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re‐investigation of the primordium‐driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA‐seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA‐seq values. Additionally, peanut AHL23 (AT‐HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA‐seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.

Primordia formation is the first and most critical step in the development of fruiting bodies of edible fungi. In this study, the effects of exogenous ascorbic acid (ASA) on the Pleurotus ostreatus mycelia growth and primordia formation were researched and the results showed that the growth rate of P. ostreatus mycelia was accelerated and the time of primordia formation was advanced. The protein content and ascorbate oxidase (AAO) activity analysis showed that with the increase of ASA concentration, the protein content of mycelia first decreased and then increased, and in a certain concentration range, exogenous ASA could significantly promote the activity of AAO. Further expression analysis of the development regulating genes (Pofst3 and Pofst4) as well as blue light receptor coding genes (PoWC-1 and PoWC-2) showed the expression levels of those four genes all changed after the exogenous ASA addition, which indicated that the expression changes of PoWC-1 and PoWC-2, two key genes in the light morphogenesis, might affect the expression levels of development regulating genes Pofst3 and Pofst4, so as to lead to the formation of primordia in advance.

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia., In soybean, nodule primordium formation involves GmPIN1-mediated polar auxin transport within primordium cells, and nodule enlargement involves the collaboration of GmPIN9d and GmPIN1-dependent auxin transport within nodule vasculature.

Plant leaves display considerable variation in shape. Here, we introduce key aspects of leaf development, focusing on the morphogenetic basis of leaf shape diversity. We discuss the importance of the genetic control of the amount, duration, and direction of cellular growth for the emergence of leaf form. We highlight how the combined use of live imaging and computational frameworks can help conceptualize how regulated cellular growth is translated into different leaf shapes. In particular, we focus on the morphogenetic differences between simple and complex leaves and how carnivorous plants form three-dimensional insect traps. We discuss how evolution has shaped leaf diversity in the case of complex leaves, by tinkering with organ-wide growth and local growth repression, and in carnivorous plants, by modifying the relative growth of the lower and upper sides of the leaf primordium to create insect-digesting traps.

The life cycle of the nematode Cosmocerca ornata (Dujardin, 1845)—a parasite of amphibians—was studied for the first time. The experimental infection of yearlings of the marsh frog Pelophylax ridibundus infected by larvae of C. ornata was performed. The development stages of parasites in the host were studied. It has been revealed that amphibians are infected with infectious larvae (J3) of nematodes in the surface-water layer by the penetration of parasites through eyes onto the conjunctiva under the lower eyelid, where the larvae undergo the third and fourth molting. Then the parasites migrate into the oropharyngeal cavity and through the digestive tract to the place of constant localization: the posterior part of the intestine. Morphological descriptions and original drawings of J3 and J4 juveniles, molting juveniles, and adults of C. ornata are given. The development stages of the juveniles are diagnosed by the body size, location of the genital primordium, and forming copulative structure. The sex of the nematodes can be identified at the third molt stage. Mature nematodes differ from juveniles by the presence of copulative organs: the vulva in adult females and spicules in adult males. The time of the development stages of C. ornata and the total life span of the parasite in the host is determined. The effect of ambient temperature on the period of development of nematodes in amphibians is established.

Arabidopsis ANT and AIL6 directly regulate genes involved in different aspects of early flower development including genes that specify floral organ identity and those that regulate growth and vascular development., Arabidopsis flower primordia give rise to organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular mechanisms by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT- and AIL6-binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT and/or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include class B and C floral homeotic genes, growth regulatory genes, and genes involved in vascular development.

Premise As a leaf expands, its shape dynamically changes. Previously, we documented an allometric relationship between vein and blade area in grapevine leaves. Larger leaves have a smaller ratio of primary and secondary vein area relative to blade area compared to smaller leaves. We sought to use allometry as an indicator of leaf size and plasticity. Methods We measured the ratio of vein-to-blade area from the same 208 vines across four growing seasons (2013, 2015, 2016, and 2017). Matching leaves by vine and node, we analyzed the correlation between the size and shape of grapevine leaves as repeated measures with climate variables across years. Results The proportion of leaf area occupied by vein and blade exponentially decreased and increased, respectively, during leaf expansion making their ratio a stronger indicator of leaf size than area itself. Total precipitation and leaf wetness hours of the previous year but not the current showed strong negative correlations with vein-to-blade ratio, whereas maximum air temperature from the previous year was positively correlated. Conclusions Our results demonstrate that vein-to-blade ratio is a strong allometric indicator of leaf size and plasticity in grapevines measured across years. Grapevine leaf primordia are initiated in buds the year before they emerge, and we found that total precipitation and maximum air temperature of the previous growing season exerted the largest statistically significant effects on leaf morphology. Vein-to-blade ratio is a promising allometric indicator of relationships between leaf morphology and climate, the robustness of which should be explored further.

Phytohormones participate in various processes over the course of a plant's lifecycle. In addition to the five classical phytohormones (auxins, cytokinins, gibberellins, abscisic acid, and ethylene), phytohormones such as brassinosteroids, jasmonic acid, salicylic acid, strigolactones, and peptides also play important roles in plant growth and stress responses. Given the highly interconnected nature of phytohormones during plant development and stress responses, it is challenging to study the biological function of a single phytohormone in isolation. In the current Review, we describe the combined functions and signaling cascades (especially the shared points and pathways) of various phytohormones in leaf development, in particular, during leaf primordium initiation and the establishment of leaf polarity and leaf morphology as well as leaf development under various stress conditions. We propose a model incorporating the roles of multiple phytohormones in leaf development and stress responses to illustrate the underlying combinatorial signaling pathways. This model provides a reference for breeding stress-resistant crops.

StCDPK2 is an early player in the salt stress response in potato plants; its overexpression promoted ROS scavenging, chlorophyll stability, and the induction of stress-responsive genes conferring tolerance to salinity. The salinity of soils affects plant development and is responsible for great losses in crop yields. Calcium-dependent protein kinases (CDPKs) are sensor–transducers that decode Ca2+ signatures triggered by abiotic stimuli and translate them into physiological responses. Histochemical analyses of potato plants harboring StCDPK2 promoter fused to the reporter gene β-glucuronidase (ProStCDPK2:GUS) revealed that GUS activity was high in the leaf blade and veins, it was restricted to root tips and lateral root primordia, and was observed upon stolon swelling. Comparison with ProStCDPK1:GUS and ProStCDPK3:GUS plants revealed their differential activities in the plant tissues. ProStCDPK2:GUS plants exposed to high salt presented enhanced GUS activity in roots which correlated with the numerous stress-responsive sites predicted in its promoter sequence. Moreover, StCDPK2 expression increased in in vitro potato plants after 2 h of high salt exposure and in greenhouse plants exposed to a dynamic stress condition. As inferred from biometric data and chlorophyll content, plants that overexpress StCDPK2 were more tolerant than wild-type plants when exposed to high salt. Overexpressing plants have a more efficient antioxidant system; they showed reduced accumulation of peroxide and higher catalase activity under salt conditions, and enhanced expression of WRKY6 and ERF5 transcription factors under control conditions. Our results indicate that StCDPK2 is an early player in the salt stress response and support a positive correlation between StCDPK2 overexpression and tolerance towards salt stress.

A common morphological feature of typical angiosperms is the patterning of lateral organs along primary axes of asymmetry—a proximodistal, a mediolateral, and an adaxial–abaxial axis. Angiosperm leaves usually have distinct adaxial–abaxial identity, which is required for the development of a flat shape. By contrast, many unifacial leaves, consisting of only the abaxial side, show a flattened morphology. This implicates a unique mechanism that allows leaf flattening independent of adaxial–abaxial identity. In this study, we report a role for auxin in outgrowth of unifacial leaves. In two closely related unifacial-leaved species of Juncaceae, Juncus prismatocarpus with flattened leaves, and Juncus wallichianus with transversally radialized leaves, the auxin-responsive gene GLYCOSIDE HYDROLASE3 displayed spatially different expression patterns within leaf primordia. Treatment of J. prismatocarpus seedlings with exogenous auxin or auxin transport inhibitors, which disturb endogenous auxin distribution, eliminated leaf flatness, resulting in a transversally radialized morphology. These treatments did not affect the radialized morphology of leaves of J. wallichianus. Moreover, elimination of leaf flatness by these treatments accompanied dysregulated expression of genetic factors needed to specify the leaf central-marginal polarity in J. prismatocarpus. The findings imply that lamina outgrowth of unifacial leaves relies on proper placement of auxin, which might induce initial leaf flattening and subsequently act to specify leaf polarity, promoting further flattening growth of leaves.

The current study was designed to describe the prenatal developmental stages of the rabbit tongue. Thirty-five embryos of prenatal ages (E16, E19, E23, E25, E28, and Postnatal) were obtained from New Zealand white (NZW) rabbit. The primordia of the tongue were observed on the foundation as early as day 16 prenatal age (E16). The first primordia of lingual papillae were observed that including the primordia of circumvallate papillae at day 19 prenatal (E19). Thickenings of lingual epithelium developed at 19 day prenatal of embryo. At day 23-25 pc, the presence of primordia of foliate papillae was shown in addition to primordia of the fungiform and filiform papillae were found. The development of the final shape of the tongue is a longstanding process that occurs during the prenatal and postnatal life. The first primordia of gustatory papillae appear, and only later primordia of mechanical papillae are formed. In conclusion, the morphogenesis of the tongue and its lingual papillae discussed its herbivorous nature of feeding.

We identified a RING-type E3 ligase (TaBAH1) protein in winter wheat that targets TaSAHH1 for degradation and might be involved in primordia development by regulating targeted protein degradation. Grain yield per spike in wheat (Triticum aestivum), is mainly determined prior to flowering during mature primordia development; however, the genes involved in primordia development have yet to be characterized. In this study, we demonstrated that, after vernalization for 50 days at 4 °C, there was a rapid acceleration in primordia development to the mature stages in the winter wheat cultivars Keumgang and Yeongkwang compared with the Chinese Spring cultivar. Although Yeongkwang flowers later than Keumgang under normal condition, it has the same heading time and reaches the WS9 stage of floral development after vernalization for 50 days. Using RNA sequencing, we identified candidate genes associated with primordia development in cvs. Keumgang and Yeongkwang, that are differentially expressed during wheat reproductive stages. Among these, the RING-type E3 ligase TaBAH1 (TraesCS5B01G373000) was transcriptionally upregulated between the double-ridge (WS2.5) stage and later stages of floret primordia development (WS10) after vernalization. Transient expression analysis indicated that TaBAH1 was localized to the plasma membrane and nucleus and was characterized by self-ubiquitination activity. Furthermore, we found that TaBAH1 interacts with TaSAHH1 to mediate its polyubiquitination and degradation through a 26S proteasomal pathway. Collectively, the findings of this study indicate that TaBAH1 might play a prominent role in post-vernalization floret primordia development.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

The stereotyped dimensions of animal bodies and their component parts result from tight constraints on growth. Yet, the mechanisms that stop growth when organs reach the right size are unknown. Growth of the Drosophila wing—a classic paradigm—is governed by two morphogens, Decapentaplegic (Dpp, a BMP) and Wingless (Wg, a Wnt). Wing growth during larval life ceases when the primordium attains full size, concomitant with the larval-to-pupal molt orchestrated by the steroid hormone ecdysone. Here, we block the molt by genetically dampening ecdysone production, creating an experimental paradigm in which the wing stops growing at the correct size while the larva continues to feed and gain body mass. Under these conditions, we show that wing growth is limited by the ranges of Dpp and Wg, and by ecdysone, which regulates the cellular response to their signaling activities. Further, we present evidence that growth terminates because of the loss of two distinct modes of morphogen action: 1) maintenance of growth within the wing proper and 2) induced growth of surrounding “pre-wing” cells and their recruitment into the wing. Our results provide a precedent for the control of organ size by morphogen range and the hormonal gating of morphogen action.

In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect organogenesis using leaf segments cut from seedlings of in vitro-germinated seeds. We obtained 85% callus induction from the leaf segments within 4–5 weeks when grown on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP, 1.0 mg/L) and 1-naphthaleneacetic acid (NAA, 0.5 mg/L). We found that MS media supplemented with Kn (1.0 mg/L) in combination with NAA (1.0 mg/L) and BAP (1.0 mg/L) combined with NAA (0.5 mg/L) were the most effective in promoting shoot regeneration, yielding plantlets with 6.72 and 6.48 shoots per culture, respectively. When cultured on PGR-free half-strength MS medium, regenerated plants developed root systems with an average 11.0 roots per shoot cluster and an average length of 36.14 mm at 9 weeks. During acclimatization, regenerated plantlets showed 96.4% survival and exhibited normal growth characteristics and morphology. We also made an attempt to directly regenerate A. cochinchinensis from shoot apices but it was futile. Histological analyses revealed the presence of crystal idioblasts in young leaves from the early stages of leaf differentiation. The leaf-based plant regeneration technique developed herein could be employed for large-scale propagation of the plants over a short time period, thereby substantially contributing to the germplasm preservation and rapid propagation of A. cochinchinensis. A repeatable in vitro micropropagation protocol for Asparagus cochinchinensis was developed based on indirect organogenesis. Histological analysis revealed crystal idioblasts for the first time in leaf primordia of this species.

International audience; Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family.In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling.We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning.Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).

An ideal leaf shape is beneficial to the yield of rice. Molecular understanding of the leaf primordia and polarity establishment plays a significant role in exploring the genetic regulatory network of leaf morphogenesis. In recent years, researchers have cloned an array of coding genes and a few non-coding small RNAs involved in rice leaf development through regulating the development of leaf primordia, vascular bundles, sclerenchyma cells, bulliform cells, cell walls and epidermis cells. These genes and their interactions play critical roles in rice leaf development through the determination and regulatory role in gene expression, and their coordination with other genetic networks or signal pathways. But the relationship among these genes is poorly defined and the underlying network is still unclear. In this review, we introduced the regulatory pathways of leaf primordium development and leaf polarity establishment, mainly the relationship between cell development mechanism and leaf polarity establishment, focusing on how leaf tissue affects leaf shape. Hopefully, the regulation network reviewed here has immediate implications for future research and genomic design breeding.

Coprinopsis cinerea has seven homologs of the Aspergillus nidulans transcription factor NsdD. Of these, CcNsdD1 and CcNsdD2 from C. cinerea show the best identities of 62 and 50% to A. nidulans NsdD, respectively. After 4 days of constant darkness cultivation, CcnsdD2, but not CcnsdD1, was upregulated on the first day of light/dark cultivation to induce fruiting bodies, and overexpression of CcnsdD2, but not CcnsdD1, produced more fruiting bodies under a light/dark rhythm. Although single knockdown of CcnsdD2 did not affect fruiting body production due to upregulation of its homolog CcnsdD1, the double-knockdown CcNsdD1/NsdD2-RNAi transformant showed defects in fruiting body formation under a light/dark rhythm. Knockdown of CcnsdD1/nsdD2 led to the differentiation of primary hyphal knots into sclerotia rather than secondary hyphal knots under a light/dark rhythm, similar to the differentiation of primary hyphal knots into sclerotia of the wild-type strain under darkness. The CcNsdD2-overexpressing transformant produced more primary hyphal knots, secondary hyphal knots, and fruiting bodies under a light/dark rhythm but only more primary hyphal knots and sclerotia under darkness. RNA-seq revealed that some genes reported previously to be involved in formation of hyphal knots and primordia, cyclopropane-fatty-acyl-phospholipid synthases cfs1-3, galectins cgl1-3, and hydrophobins hyd1-3 were downregulated in the CcNsdD1/NsdD2-RNAi transformant compared to the mock transformant. ChIP-seq and electrophoretic mobility shift assay demonstrated that CcNsdD2 bound to promoter regulatory sequences containing a GATC motif in cfs1, cfs2, cgl1, and hyd1. A molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions is proposed.

Vegetative propagules play various important roles in lichen biology. We cultured soredia of Cladonia lichens in vitro and obtained three noteworthy results. Firstly, soredia are a beneficial source for the isolation of lichen symbionts. The mycobiont was obtained from 66% and the photobiont from 67% of the cultured soredia that were not contaminated. Secondly, the development of soredia followed a previously recognized pattern, arachnoid stage – soredium field – primordium, but a thalline structure was not achieved. We suggest that thallus formation in vitro is a question of favourable environmental factors, not partners compatibility. Finally, we discovered that fungi, other than the mycobiont, as well as airborne contaminants are dispersed together with lichen soredia. This is the first-ever report of such a phenomenon. The possible ecological consequences are discussed. Cystobasidiomycete yeasts were found among these fungi. We isolated representatives of three different lineages from a single thallus suggesting a low specificity for this association.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

The plant-specific NAC transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) functions in xylem vessel cell differentiation in Arabidopsis thaliana. To identify novel factors regulating xylem vessel cell differentiation, we previously performed ethyl methanesulfonate mutagenesis of a transgenic 35S::VND7-VP16-GR line in which VND7 activity can be induced post-translationally by glucocorticoid treatment. We successfully isolated mutants that fail to form ectopic xylem vessel cells named seiv (suppressor of ectopic vessel cell differentiation induced by VND7) mutants. Here, we isolated eight novel dominant seiv mutants: seiv2 to seiv9. In these seiv mutants, ectopic xylem vessel cell differentiation was inhibited in aboveground but not underground tissues. Specifically, the shoot apices of the mutants, containing shoot apical meristems and leaf primordia, completely lacked ectopic xylem vessel cells. In these mutants, the VND7-induced upregulation of downstream genes was reduced, especially in shoots compared to roots. However, endogenous xylem vessel cell formation was not affected in the seiv mutants. Therefore, the seiv mutations identified in this study have repressive effects on cell differentiation in shoot meristematic regions, resulting in inhibited ectopic xylem vessel cell differentiation.

More than 80,000 angiosperm species produce flowers with petals fused into a corolla tube. As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower speciesMimulus lewisii. Loss-of-function mutations in theM. lewisiiorthologs ofARGONAUTE7andSUPPRESSOR OF GENE SILENCING 3cause a dramatic decrease in abundance ofTAS3-derived small RNAs and a moderate up-regulation ofAUXIN RESPONSE FACTOR 3(ARF3) andARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. By using an auxin reporter construct, we discovered that auxin distribution is continuous along the petal primordium base and the inter-primordial region during the critical stage of corolla tube formation in the wild-type, and that this auxin distribution is much weaker and more restricted in the mutant. Together, these results suggest a new conceptual model highlighting the central role of auxin directed synchronized growth of the petal primordium base and the inter-primordial region in corolla tube formation.

The development of flowers and floral organs is directed by intricate genetic programmes, many aspects of which appear to be shared among angiosperms. Early acting genes establish floral meristem identity in flower primordia initiated at the periphery of the inflorescence meristem. Later, floral organ primordia arise at precise positions within these floral meristems and take on one of the four distinct identities (sepals, petals, stamens and carpels). The ABCE model, supported by both molecular and genetic experiments in Arabidopsis, explains how a small number of regulatory genes (called floral homeotic genes or floral organ identity genes) act in different combinations to specify these different organ types. The floral organ identity genes encode transcription factors that form distinct higher order protein complexes in different regions of a flower primordium to control the expression of target genes responsible for organogenesis. Key Concepts Lateral organs produced by the shoot apical meristem during reproductive development acquire their identity as flowers through the action of floral meristem identity genes such as LEAFY and APETALA1. The identities of each of the four organ types of a flower (sepal, petal, stamen and carpel) is conferred by a unique combination of floral organ identity gene activities, referred to as class A, B, C and E in the ABCE model. The activities of the class A, B and C genes are restricted to particular regions within a developing flower primarily, but not exclusively, through transcriptional regulation. The MADS domain transcription factors encoded by the class A, B, C and E genes form unique tetrameric transcriptional regulatory complexes in cells of each floral whorl. The transcriptional regulatory complexes formed by the A, B, C and E proteins regulate distinct sets of genes at different stages of flower development. Many aspects of the genetic programmes conferring floral meristem identity and floral organ identity are conserved among all angiosperms. Keywords: flower development; homeotic genes; floral meristem identity genes; floral organ identity genes; Arabidopsis; ABCE model; quartet model

Made available in DSpace on 2020-12-12T02:41:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) New species of medicinal mushrooms have emerged over the past several decades, such as the Sun mushroom, Agaricus subrufescens. Horticultural improvements are required to shift its cultivation from small-scale local production to large-scale international production. The research reported here evaluated the agronomic behavior and the chemical characteristics of the Sun mushroom as a function of i) nutritional supplementation ii) ruffling of the casing layer and iii) the temperature management on the primordia induction and reduction of the crop cycle. Supplementation was beneficial for yield, unit mushroom weigh and decrease in time to first harvest. Supplementation improved biological efficiency with Champfood providing a yield increase of 15% over the non-supplemented compost. Among the supplements only Promycel increased the individual mushroom weight. Ruffling overall improved the yield in the 2nd and 4th flush. Already biological efficiency was greater by 21%. The highest yield harvested in any single day in the crop occurred in 3rd flush with the amount of 2.484 kg of mushrooms per m2 for the rapid induction method. Still the biological efficiency was not significantly affected by the mushroom induction temperature method. Only the fat content of the mushrooms was positively affected by the rapid induction of primordia. Champfood supplement promotes a reduction in the value of earliness and an increase of 1st flush yield. The ruffling technique provided an increase in biological efficiency due to the great number of mushrooms harvested. Rapid primordia induction allowed the crop cycle to end 3 days earlier than the slow primordia induction, providing a higher production rate. Centro de Investigación Experimentación y Servicios del Champiñón (CIES) Escuela Técnica Superior de Ingenieros Agrónomos Universidad de Castilla-La Mancha Universidade Federal de Lavras Departamento de Biologia University of Guelph Vineland Campus VinelandStation Universidade Estadual Paulista (UNESP) Faculdade de Ciências Agrárias e Tecnológicas (FCAT) Universidade Estadual Paulista (UNESP) Faculdade de Ciências Agrárias e Tecnológicas (FCAT) FAPESP: FAPESP 2018/21492-2

Interactions between primordium cells and their environment determines the self-organization of the zebrafish posterior Lateral Line primordium as it migrates under the skin from the ear to the tip of the tail forming and depositing neuromasts to spearhead formation of the posterior Lateral Line sensory system. In this review we describe how the NetLogo agent-based programming environment has been used in our lab to visualize and explore how self-generated chemokine gradients determine collective migration, how the dynamics of Wnt signaling can be used to predict patterns of neuromast deposition, and how previously defined interactions between Wnt and Fgf signaling systems have the potential to determine the periodic formation of center-biased Fgf signaling centers in the wake of a shrinking Wnt system. We also describe how NetLogo was used as a database for storing and visualizing the results of in toto lineage analysis of all cells in the migrating primordium. Together, the models illustrate how this programming environment can be used in diverse ways to integrate what has been learnt from biological experiments about the nature of interactions between cells and their environment, and explore how these interactions could potentially determine emergent patterns of cell fate specification, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.

The application of molecular breeding of eucalyptus is restricted by the difficulty of regeneration. One limitation of eucalyptus regeneration could be the production of excessive reactive oxygen species (ROS) during in vitro culture that greatly affects the shoot regeneration. NADPH oxidases, encoded by the respiratory burst oxidase homologue (rboh), and ascorbate peroxidases (APX) play an important role in the ROS metabolism. The study investigated the combined effect and preliminary mechanism on eucalyptus regeneration of N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), 6-benzylaminopurine (BAP), and dimethylthiourea (DMTU) by inoculating Eucalyptus urophylla hypocotyls on SPCa medium supplemented with 0.57 µM indole-3-acetic acid (IAA) and various ratios of PBU and BAP. In the presence of 4.56 µM PBU alone, the rboh transcription levels were relatively low and the APX transcription levels were relatively high in the 2-week-old calli. However, the highest frequency of organogenic calli was induced by the combination of 3.99 µM PBU, 0.57 µM BAP and 0.57 µM IAA. DMTU, a ROS scavenger, was added in 10, 20, or 30 µM increments to the SPCa medium together with 3.99 µM PBU and 0.57 µM BAP to promote callus differentiation. The expression levels of all nine APX genes increased significantly in the calli following treatment with DMTU. The calli treated with 20 µM DMTU showed the highest APX transcriptional levels, which was consistent with the highest APX activity, and formed the most organogenic calli. During the subsequent process of adventitious shoot formation, the transcription levels of rboh1 showed a descending pattern. However, the transcription levels of rboh4, rboh5, and rboh6 showed an ascending pattern and peaked in the calli with visible shoot primordia. Specifically, the expression of rboh5 and rboh6 was 203.2 times and 573.9 times higher than that in the 2-week-old calli, respectively. Additionally, the calli with visible shoot primordia showed the highest H2O2 content. The results implied that control of oxidative stress by a combination of PBU, BAP, and DMTU enhances adventitious shoot formation in E. urophylla by affecting the expression of the rboh and APX genes and regulating ROS metabolism. The results would be helpful for an efficient regeneration protocol for eucalyptus. Cooperation of PBU, BAP, and DMTU enhances adventitious shoot formation in Eucalyptus urophylla by affecting the expression of rboh and APX genes.

Summary VERNALIZATION2 (VRN2), an angiosperm‐specific subunit of the polycomb repressive complex 2 (PRC2), is an oxygen (O2)‐regulated target of the PCO branch of the PRT6 N‐degron pathway of ubiquitin‐mediated proteolysis. How this post‐translational regulation coordinates VRN2 activity remains to be fully established.Here we use Arabidopsis thaliana ecotypes, mutants and transgenic lines to determine how control of VRN2 stability contributes to its functions during plant development.VRN2 localizes to endogenous hypoxic regions in aerial and root tissues. In the shoot apex, VRN2 differentially modulates flowering time dependent on photoperiod, whilst its presence in lateral root primordia and the root apical meristem negatively regulates root system architecture. Ectopic accumulation of VRN2 does not enhance its effects on flowering, but does potentiate its repressive effects on root growth. In late‐flowering vernalization‐dependent ecotypes, VRN2 is only active outside meristems when its proteolysis is inhibited in response to cold exposure, as its function requires concomitant cold‐triggered increases in other PRC2 subunits and cofactors.We conclude that the O2‐sensitive N‐degron of VRN2 has a dual function, confining VRN2 to meristems and primordia, where it has specific developmental roles, whilst also permitting broad accumulation outside of meristems in response to environmental cues, leading to other functions., See also the Editorial on this article by Sasidharan et al., 229: 5–7.

In this study, embryos of Sepiella japonica from eye primordium formation to the larval growing stage were collected and used for RNA-Seq analysis. A total of 183,542,186 clean reads were assembled...

Receptor-like kinases (RLKs) are important for plant growth, development and defense responses. The S-receptor protein kinases (SRKs), which represent an RLK subfamily, control the self- incompatibility among Brassica species. However, little information is available regarding SRK functions in rice. We identified a gene OsSRK1 encoding an atypical SRK. The transcript of OsSRK1 was induced by abscisic acid (ABA), salt and polyethylene glycol. OsSRK1 localized to the plasma membrane and cytoplasm. Leaf width was increased in OsSRK1-overexpression (OsSRK1-OX) transgenic rice plants, likely because of an increase in cell number per leaf. Furthermore, the expression levels of OsCYCA3-1 and OsCYCD2-1, which encode positive regulators of cell division, were up-regulated in leaf primordium of OsSRK1-OX rice plants relative to those in wild type. Meanwhile, the expression level of OsKRP1, which encodes cell cycle inhibitor, was down-regulated in the OsSRK1-OX plants. Therefore, it is deduced that OsSRK1 regulates leaf width by promoting cell division in the leaf primordium. Additionally, OsSRK1-OX plants exhibited enhanced ABA sensitivity and salt tolerance compared with wild type. These results suggest that OsSRK1 plays important roles in leaf development and salt responses in rice. Keywords: rice, S-receptor-like protein kinase, OsSRK1, salt tolerance, leaf width, cell division

Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induction, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene, which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction. The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression increased in the apical bud when corms are storage at 9-10 ºC, thus inhibiting flower induction. Additionally, comparison of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angle towards the available water. Our observations reveal how the external hydrological environment regulates lateral root morphogenesis. Arabidopsis lateral root radial emergence is spatially constrained by the dual xylem poles in the main root. However, the asymmetric presence of water can modify the orientation of lateral roots at a very early stage.

An overview of the gene and hormone regulatory network involved in determining ovule number and gynoecium size, two fundamental processes that affect seed yield., Angiosperms form the largest group of land plants and display an astonishing diversity of floral structures. The development of flowers greatly contributed to the evolutionary success of the angiosperms as they guarantee efficient reproduction with the help of either biotic or abiotic vectors. The female reproductive part of the flower is the gynoecium (also called pistil). Ovules arise from meristematic tissue within the gynoecium. Upon fertilization, these ovules develop into seeds while the gynoecium turns into a fruit. Gene regulatory networks involving transcription factors and hormonal communication regulate ovule primordium initiation, spacing on the placenta, and development. Ovule number and gynoecium size are usually correlated and several genetic factors that impact these traits have been identified. Understanding and fine-tuning the gene regulatory networks influencing ovule number and pistil length open up strategies for crop yield improvement, which is pivotal in light of a rapidly growing world population. In this review, we present an overview of the current knowledge of the genes and hormones involved in determining ovule number and gynoecium size. We propose a model for the gene regulatory network that guides the developmental processes that determine seed yield.

The flower of Zingiber zerumbet is characterized by a distinctive labellum, a highly modified floral organ believed to be formed by the fusion of several infertile members of the androecial whorl (staminodes). Across the Zingiberaceae, the number of staminodes involved in labellum formation varies from two to four, and these are reflected in the number of lobes that comprise the mature labellum. Research on the flower development in Zingiberaceae has been limited to species with either no labellum lobes or species displaying a bilobed labellum. Zingiber zerumbet is a representative of the genus with a three-lobed labellum, and its flower development remains poorly understood at both morphological and molecular levels. This study aims to give a comprehensive description of its flower development and to identify potential genes related to flower development using morphological and genetic characterization. Our results show that floral organ initiation is sequential with the sepal whorl initiating first, followed by petal and inner androecium together, followed by outer androecium, and finally the initiation of the inferior gynoecium. The three-lobed labellum comprises four androecial members: Two abaxial inner androecial members fuse to form the single central lobe, and two adaxial outer androecial members individually form the two lateral lobes of the labellum. Two developmental stages (floral primordium and organ-differentiated flowers) were selected for transcriptome sequencing. Two-thousand and seventy-five transcription factors were identified. Seven boundary genes and seven organ-specific genes were also discovered. Our study provides fundamental information for further studies on the molecular mechanisms of flower development and evolution across the Zingiberaceae.

Lateral roots originate from initial cells deep within the main root and must emerge through several overlying layers. Lateral root emergence requires the outgrowth of the new primordium (LRP) to coincide with the timely separation of overlying root cells, a developmental program coordinated by the hormone auxin. Here, we report that in Arabidopsis thaliana roots, auxin controls the spatiotemporal expression of the plasmodesmal regulator PDLP5 in cells overlying LRP, creating a negative feedback loop. PDLP5, which functions to restrict the cell-to-cell movement of signals via plasmodesmata, is induced by auxin in cells overlying LRP in a progressive manner. PDLP5 localizes to plasmodesmata in these cells and negatively impacts organ emergence as well as overall root branching. We present a model, incorporating the spatiotemporal expression of PDLP5 in LRP-overlying cells into known auxin-regulated LRP-overlying cell separation pathways, and speculate how PDLP5 may function to negatively regulate the lateral root emergence process., Auxin promotes lateral root emergence from pericycle cells in the root vasculature. Here the authors show that induction of the plasmodesmal regulator PDLP5 during lateral root emergence restricts the spatial scope of auxin signaling to the cells overlying the primordia.

The development of root nodules leads to an increased auxin response in early nodule primordia, which is mediated by changes in acropetal auxin transport in some legumes. Here, we investigated the role of root basipetal auxin transport during nodulation. Rhizobia inoculation significantly increased basipetal auxin transport in both Medicago truncatula and Lotus japonicus. In M. truncatula, this increase was dependent on functional Nod factor signalling through NFP, NIN, and NSP2, as well as ethylene signalling through SKL. To test whether increased basipetal auxin transport is required for nodulation, we examined a loss-of-function mutant of the M. truncatula PIN2 gene. The Mtpin2 mutant exhibited a reduction in basipetal auxin transport and an agravitropic phenotype. Inoculation of Mtpin2 roots with rhizobia still led to a moderate increase in basipetal auxin transport, but the mutant nodulated normally. No clear differences in auxin response were observed during nodule development. Interestingly, inoculation of wild-type roots increased lateral root numbers, whereas inoculation of Mtpin2 mutants resulted in reduced lateral root numbers compared with uninoculated roots. We conclude that the MtPIN2 auxin transporter is involved in basipetal auxin transport, that its function is not essential for nodulation, but that it plays an important role in the control of lateral root development.

Key message Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Abstract Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

We previously identified the FLOWERING LOCUS C (FLC)-like gene, a MADS-box transcription factor gene that belongs to Arabidopsis thaliana L. FLC clade, in apple (Malus $\times$ domestica Borkh.), and its expression in dormant flower buds is positively correlated with cumulative cold exposure. To elucidate the role of the MdFLC-like in the dormancy process and flower development, we first characterized the phenotypes of MdFLC-like overexpressing lines with the Arabidopsis Columbia-0 background. The overexpression of MdFLC-like significantly delayed the bolting date and reduced the plant size, but it did not significantly affect the number of rosette leaves or flower organ formation. Thus, MdFLC-like may affect vegetative growth and development rather than flowering when expressed in Arabidopsis, which is not like Arabidopsis FLC that affects development of flowering. We compared seasonal expression patterns of MdFLC-like in low-chill ‘Anna’ and high-chill ‘Fuji’ and ‘Tsugaru’ apples collected from trees grown in a cold winter region in temperate zone and found an earlier upregulation in ‘Anna’ compared with ‘Fuji’ and ‘Tsugaru’. Expression patterns were also compared in relation to developmental changes in the flower primordia during the chilling accumulation period. Overall, MdFLC-like was progressively upregulated during flower primordia differentiation and development in autumn to early winter and reached a maximum expression level at around the same time as the genotype-dependent chilling requirements were fulfilled in high-chill cultivars. Thus, we hypothesize MdFLC-like may be upregulated in response to cold exposure and flower primordia development during the progress of endodormancy. Our study also suggests MdFLC-like may have a growth-inhibiting function during the end of endodormancy and ecodormancy when the temperature is low and unfavorable for rapid bud outgrowth.

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding.

In dicot plants, the process by which undifferentiated plastids, termed proplastids, differentiate into mature functional chloroplasts begins in the shoot apical meristem (SAM) and young leaf primordia, and continues along leaf development. In this work, we followed initial chloroplast biogenesis in cells of the SAM in Arabidopsis, during the early stages of germination, using chlorophyll fluorescence as a marker. We found that cells bound to form the SAM in the mature seed embryo lack chlorophyll, while plastids in the rest of the embryo cells are somewhat developed. The initial appearance of chlorophyll in the SAM occurred two days after the onset of germination, was not expedited by higher light intensities, and required a light period of between five to ten hours within these two days. In addition, we found that biogenesis of chloroplasts occurred only in the upper layer of the SAM, as opposed to the two central subtending cell layers. This pattern was maintained, mirroring the developmental status of plastids in the mature vegetative SAM. The work also presents another model for studying proplastid-to-chloroplast development, in which differentiation can be followed in SAM cells in a defined time-wise fashion.

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

Leaf development is crucial to establish the photosynthetic competency of plants. It is a process that requires coordinated changes in cell number and differentiation, transcriptomes, metabolomes and physiology. However, despite the importance of leaf formation for our major crops, early developmental processes for rice have not been comprehensively described. Here we detail the temporal developmental trajectory of early rice leaf development and connect morphological changes to metabolism. In particular, a developmental index based on the patterning of epidermal differentiation visualised by electron microscopy enabled high resolution staging of early growth for single primordium metabolite profiling. These data demonstrate that a switch in the constellation of tricarboxylic acid (TCA) cycle metabolites defines a narrow window towards the end of the P3 stage of leaf development. Taken in the context of other data in the literature, our results substantiate that this phase of rice leaf growth, equivalent to a change of primordium length from around 5 to 7.5 mm, defines a major shift in rice leaf determination towards a photosynthetically defined structure. We speculate that efforts to engineer rice leaf structure should focus on the developmental window prior to these determining events.HighlightRice leaves undergo a shift in fundamental metabolism during a very early and narrow developmental window which co-incides with them acquiring the ability to capture light for photosynthesis

Background Liriodendron chinense is a distinctive ornamental tree species due to its unique leaves and tulip-like flowers. The discovery of genes involved in leaf development and morphogenesis is critical for uncovering the underlying genetic basis of these traits. Genes in the AP2/ERF family are recognized as plant-specific transcription factors that contribute to plant growth, hormone-induced development, ethylene response factors, and stress responses. Results In this study, we identified 104 putative AP2/ERF genes in the recently released L. chinense genome and transcriptome database. In addition, all 104 genes were grouped into four subfamilies, the AP2, ERF, RAV, and Soloist subfamilies. This classification was further supported by the results of gene structure and conserved motif analyses. Intriguingly, after application of a series test of cluster analysis, three AP2 genes, LcERF 94, LcERF 96, and LcERF 98, were identified as tissue-specific in buds based on the expression profiles of various tissues. These results were further validated via RT-qPCR assays and were highly consistent with the STC analysis. We further investigated the dynamic changes of immature leaves by dissecting fresh shoots into seven discontinuous periods, which were empirically identified as shoot apical meristem (SAM), leaf primordia and tender leaf developmental stages according to the anatomic structure. Subsequently, these three candidates were highly expressed in SAM and leaf primordia but rarely in tender leaves, indicating that they were mainly involved in early leaf development and morphogenesis. Moreover, these three genes displayed nuclear subcellular localizations through the transient transformation of tobacco epidermal cells. Conclusions Overall, we identified 104 AP2/ERF family members at the genome-wide level and discerned three candidate genes that might participate in the development and morphogenesis of the leaf primordium in L. chinense.

Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. Caenorhabditis elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female distal tip cells (fDTCs), while the anchor cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie the evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that hlh-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.

Background Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. Methods A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. Results At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. Conclusions A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

Fungi have been reported to be the dominant eukaryotic group in anoxic sub-seafloor sediments, but how fungi subsist in the anoxic sub-marine sedimental environment is rarely understood. Our previous study demonstrated that the fungus, Schizophyllum commune 20R-7-F01 isolated from a ~2 km sediment below the seafloor, can grow and produce primordia in the complete absence of oxygen with enhanced production of branched-chain amino acids (BCAAs), but the primordia cannot be developed into fruit bodies without oxygen. Here, we present the individual and synergistic effects of oxygen and BCAAs on the fruit-body development of this strain. It was found that the fungus required a minimum oxygen concentration of 0.5% pO2 to generate primordia and 1% pO2 to convert primordia into mature fruit body. However, if BCAAs (20 mM) were added to the medium, the primordium could be developed into fruit body at a lower oxygen concentration up to 0.5% pO2 where genes fst4 and c2h2 playing an important role in compensating oxygen deficiency. Moreover, under hypoxic conditions, the fungus showed an increase in mitochondrial number and initiation of auto-phagocytosis. These findings suggest that the fruit-body formation of S. commune may have multiple mechanisms, including energy and amino acid metabolism in response to oxygen concentrations.