Auxin Biosynthesis Research Articles

The Mediator complex subunit MED25 interacts with HDA9 and PIF4 to regulate thermomorphogenesis.

Thermomorphogenesis is, among other traits, characterized by enhanced hypocotyl elongation due to the induction of auxin biosynthesis genes like YUCCA8 by transcription factors, most notably PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Efficient binding of PIF4 to the YUCCA8 locus under warmth depends on HISTONE DEACETYLASE 9 (HDA9) activity, which mediates histone H2A.Z depletion at the YUCCA8 locus. However, HDA9 lacks intrinsic DNA-binding capacity, and how HDA9 is recruited to YUCCA8, and possibly other PIF4-target sites, is currently not well understood. The Mediator complex functions as a bridge between transcription factors bound to specific promoter sequences and the basal transcription machinery containing RNA polymerase II. Mutants of Mediator component Mediator25 (MED25) exhibit reduced hypocotyl elongation and reduced expression of YUCCA8 at 27°C. In line with a proposed role for MED25 in thermomorphogenesis in Arabidopsis (Arabidopsis thaliana), we demonstrated an enhanced association of MED25 to the YUCCA8 locus under warmth and interaction of MED25 with both PIF4 and HDA9. Genetic analysis confirmed that MED25 and HDA9 operate in the same pathway. Intriguingly, we also showed that MED25 destabilizes HDA9 protein. Based on our findings, we propose that MED25 recruits HDA9 to the YUCCA8 locus by binding to both PIF4 and HDA9.

Synergistic effects of nitrogen metabolites on auxin regulating plant growth and development.

Nitrogen is one of the important nutrients required for plant growth and development. There is increasing evidences that almost all types of nitrogen metabolites affect, at least to some extent, auxin content and/or signaling in plants, which in turn affects seed germination, plant root elongation, gravitropism, leaf expansion and floral transition. This opinion focuses on the roles of nitrogen metabolites, , , tryptophan and NO and their synergistic effects with auxin on plant growth and development. Nitrate reductase (NR) converts nitrate into nitrite, and was roughly positive-correlated with the root auxin level, suggesting a crosstalk between nitrate signaling and auxin signaling. Abscisic Acid Responsive Element Binding Factor 3 (AFB3) and Tryptophan Aminotransferase of Arabidopsis 1 (TAA1) are also the key enzymes involved in nitrogen metabolite-regulated auxin biosynthesis. Recent advances in the crosstalk among , , tryptophan and NO in regulation to NR, AFB3 and TAA1 are also summarized.

Abscisic acid promotes auxin biosynthesis to inhibit primary root elongation in rice.

Soil compaction is a global problem causing inadequate rooting and poor yield in crops. Accumulating evidence indicates that phytohormones coordinately regulate root growth via regulating specific growth processes in distinct tissues. However, how abscisic acid (ABA) signaling translates into auxin production to control root growth during adaptation to different soil environments is still unclear. In this study, we report that ABA has biphasic effects on primary root growth in rice (Oryza sativa) through an auxin biosynthesis-mediated process, causing suppression of root elongation and promotion of root swelling in response to soil compaction. We found that ABA treatment induced the expression of auxin biosynthesis genes and auxin accumulation in roots. Conversely, blocking auxin biosynthesis reduced ABA sensitivity in roots, showing longer and thinner primary roots with larger root meristem size and smaller root diameter. Further investigation revealed that the transcription factor basic region and leucine zipper 46 (OsbZIP46), involved in ABA signaling, can directly bind to the YUCCA8/rice ethylene-insensitive 7 (OsYUC8/REIN7) promoter to activate its expression, and genetic analysis revealed that OsYUC8/REIN7 is located downstream of OsbZIP46. Moreover, roots of mutants defective in ABA or auxin biosynthesis displayed the enhanced ability to penetrate compacted soil. Thus, our results disclose the mechanism in which ABA employs auxin as a downstream signal to modify root elongation and radial expansion, resulting in short and swollen roots impaired in their ability to penetrate compacted soil. These findings provide avenues for breeders to select crops resilient to soil compaction.

Comparative Transcriptome and Co-Expression Network Analyses Reveal the Molecular Mechanism of Calcium-Deficiency-Triggered Tipburn in Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).

Chinese cabbage tipburn is characterized by the formation of necrotic lesions on the margin of leaves, including on the insides of the leafy head. This physiological disorder is associated with a localized calcium deficiency during leaf development. However, little information is available regarding the molecular mechanisms governing Ca-deficiency-triggered tipburn. This study comprehensively analysed the transcriptomic comparison between control and calcium treatments (CK and 0 mM Ca) in Chinese cabbage to determine its molecular mechanism in tipburn. Our analysis identified that the most enriched gene ontology (GO) categories are photosynthesis, thylakoid and cofactor binding. Moreover, the KEGG pathway was most enriched in photosynthesis, carbon metabolism and carbon fixation. We also analyzed the co-expression network by functional categories and identified ten critical hub differentially expressed genes (DEGs) in each gene regulatory network (GRN). These DEGs might involve abiotic stresses, developmental processes, cell wall metabolism, calcium distribution, transcription factors, plant hormone biosynthesis and signal transduction pathways. Under calcium deficiency, CNX1, calmodulin-binding proteins and CMLs family proteins were downregulated compared to CK. In addition, plant hormones such as GA, JA, BR, Auxin and ABA biosynthesis pathways genes were downregulated under calcium treatment. Likewise, HATs, ARLs and TCP transcription factors were reported as inactive under calcium deficiency, and potentially involved in the developmental process. This work explores the specific DEGs' significantly different expression levels in 0 mM Ca and the control involved in plant hormones, cell wall developments, a light response such as chlorophylls and photosynthesis, transport metabolism and defence mechanism and redox. Our results provide critical evidence of the potential roles of the calcium signal transduction pathway and candidate genes governing Ca-deficiency-triggered tipburn in Chinese cabbage.

Identification of a major QTL, Parth6.1 associated with parthenocarpic fruit development in slicing cucumber genotype, Pusa Parthenocarpic Cucumber-6.

Parthenocarpy is an extremely important trait that revolutionized the worldwide cultivation of cucumber under protected conditions. Pusa Parthenocarpic Cucumber-6 (PPC-6) is one of the important commercially cultivated varieties under protected conditions in India. Understanding the genetics of parthenocarpy, molecular mapping and the development of molecular markers closely associated with the trait will facilitate the introgression of parthenocarpic traits into non-conventional germplasm and elite varieties. The F1, F2 and back-crosses progenies with a non-parthenocarpic genotype, Pusa Uday indicated a single incomplete dominant gene controlling parthenocarpy in PPC-6. QTL-seq comprising of the early parthenocarpy and non-parthenocarpic bulks along with the parental lines identified two major genomic regions, one each in chromosome 3 and chromosome 6 spanning over a region of 2.7 Mb and 7.8 Mb, respectively. Conventional mapping using F2:3 population also identified two QTLs, Parth6.1 and Parth6.2 in chromosome 6 which indicated the presence of a major effect QTL in chromosome 6 determining parthenocarpy in PPC-6. The flanking markers, SSR01148 and SSR 01012 for Parth6.1 locus and SSR10476 and SSR 19174 for Parth6.2 locus were identified and can be used for introgression of parthenocarpy through the marker-assisted back-crossing programme. Functional annotation of the QTL-region identified two major genes, Csa_6G396640 and Csa_6G405890 designated as probable indole-3-pyruvate monooxygenase YUCCA11 and Auxin response factor 16, respectively associated with auxin biosynthesis as potential candidate genes. Csa_6G396640 showed only one insertion at position 2179 in the non-parthenocarpic parent. In the case of Csa_6G405890, more variations were observed between the two parents in the form of SNPs and InDels. The study provides insight about genomic regions, closely associated markers and possible candidate genes associated with parthenocarpy in PPC-6 which will be instrumental for functional genomics study and better understanding of parthenocarpy in cucumber.

Auxin contributes to jasmonate-mediated regulation of abscisic acid signaling during seed germination in Arabidopsis.

Abscisic acid (ABA) represses seed germination and postgerminative growth in Arabidopsis thaliana. Auxin and jasmonic acid (JA) stimulate ABA function; however, the possible synergistic effects of auxin and JA on ABA signaling and the underlying molecular mechanisms remain elusive. Here, we show that exogenous auxin works synergistically with JA to enhance the ABA-induced delay of seed germination. Auxin biosynthesis, perception, and signaling are crucial for JA-promoted ABA responses. The auxin-dependent transcription factors AUXIN RESPONSE FACTOR10 (ARF10) and ARF16 interact with JASMONATE ZIM-DOMAIN (JAZ) repressors of JA signaling. ARF10 and ARF16 positively mediate JA-increased ABA responses, and overaccumulation of ARF16 partially restores the hyposensitive phenotype of JAZ-accumulating plants defective in JA signaling in response to combined ABA and JA treatment. Furthermore, ARF10 and ARF16 physically associate with ABSCISIC ACID INSENSITIVE5 (ABI5), a critical regulator of ABA signaling, and the ability of ARF16 to stimulate JA-mediated ABA responses is mainly dependent on ABI5. ARF10 and ARF16 activate the transcriptional function of ABI5, whereas JAZ repressors antagonize their effects. Collectively, our results demonstrate that auxin contributes to the synergetic modulation of JA on ABA signaling, and explain the mechanism by which ARF10/16 coordinate with JAZ and ABI5 to integrate the auxin, JA, and ABA signaling pathways.

Melatonin regulates gene expressions through activating auxin synthesis and signaling pathways.

Both melatonin and indole-3-acetic acid (IAA) are derived from tryptophan. And the most interesting and unsolved puzzle in melatonin research is that what is the relationship between melatonin and auxin? In this study, we performed transcriptome analysis with a time series method to disclose the connection of the two metabolites in soybean. Our results reveal that melatonin and IAA treatments cause substantial overlaps in gene expression changes. Common genes of melatonin and IAA treatments could be sorted into clusters with very similar expression tendency. A KEGG assay showed that exogenous applied melatonin enriched differentially expressed genes in auxin biosynthesis and signaling pathways. For details, melatonin up-regulates several YUCCA genes which participate in auxin biosynthesis; melatonin also enhances expression levels of auxin receptor coding genes, such as TIR1, AFB3 and AFB5; dozens of genes involved in auxin transport, such as AUXI and PIN, are regulated by melatonin similarly as by auxin; auxin-responsive genes, such as IAA, ARF, GH3 and SAUR-like genes, intensively respond to melatonin as well as to auxin. A DR5 promoter mediated GUS staining assay showed that low concentration of melatonin could induce auxin biosynthesis in a dosage manner, whereas high concentration of melatonin would eliminate such effect. At last, gene ontology (GO) analysis suggests that melatonin treatment has similar characteristics as auxin treatment in many processes. However, the two molecules still keep their own features respectively. For example, melatonin takes part in stress responses, while IAA treatment enriches the GO terms that related to cell growth. Taken together, exogenous applied melatonin, if not exceeds the appropriate concentration, could promote auxin responses range from biosynthesis to signaling transduction. Thus, our research is a key part to explain the auxin-like roles of melatonin in regulating plant growth.

High temporal-resolution transcriptome landscapes of maize embryo sac and ovule during early seed development

Here we provided a high temporal-resolution transcriptome atlas of maize embryo sac and ovule to reveal the gene activity dynamic during early seed development. The early maize (Zea mays) seed development is initiated from double fertilization in the embryo sac and needs to undergo a highly dynamic and complex development process to form the differentiated embryo and endosperm. Despite the importance of maize seed for food, feed, and biofuel, many regulators responsible for controlling its early development are not known yet. Here, we reported a high temporal-resolution transcriptome atlas of embryo sac and ovule based on 44 time point samples collected within the first four days of seed development. A total of 25,187 genes including 1598 transcription factors (TFs) involved in early seed development were detected. Global comparisons of the expressions of these genes revealed five distinct development stages of early seed, which are mainly related to double fertilization, asymmetric cell division of the zygote, as well as coenocyte formation, cellularization and differentiation in endosperm. We identified 3327 seed-specific genes, which more than one thousand seed-specific genes with main expressions during early seed development were newly identified here, including 859 and 186 genes predominantly expressed in the embryo sac and ovule, respectively. Combined with the published transcriptome data of seed, we uncovered the dominant auxin biosynthesis, transport and signaling related genes at different development stages and subregions of seed. These results are helpful for understanding the genetic control of early seed development.

Auxin Biosynthesis Genes in Allotetraploid Oilseed Rape Are Essential for Plant Development and Response to Drought Stress.

Crucial studies have verified that IAA is mainly generated via the two-step pathway in Arabidopsis, in which tryptophan aminotransferase (TAA) and YUCCA (YUC) are the two crucial enzymes. However, the role of the TAA (or TAR) and YUC genes in allotetraploid oilseed rape underlying auxin biosynthesis and development regulation remains elusive. In the present study, all putative TAR and YUC genes were identified in B. napus genome. Most TAR and YUC genes were tissue that were specifically expressed. Most YUC and TAR proteins contained trans-membrane regions and were confirmed to be endoplasmic reticulum localizations. Enzymatic activity revealed that YUC and TAR protein members were involved in the conversion of IPA to IAA and Trp to IPA, respectively. Transgenic plants overexpressing BnaYUC6a in both Arabidopsis and B. napus displayed high auxin production and reduced plant branch angle, together with increased drought resistance. Moreover, mutation in auxin biosynthesis BnaTARs genes by CRISPR/Cas9 caused development defects. All these results suggest the convergent role of BnaYUC and BnaTAR genes in auxin biosynthesis. Different homoeologs of BnaYUC and BnaTAR may be divergent according to sequence and expression variation. Auxin biosynthesis genes in allotetraploid oilseed rape play a pivotal role in coordinating plant development processes and stress resistance.

Polar auxin transport modulates early leaf flattening

The flattened leaf form is an important adaptation for efficient photosynthesis, and the developmental process of flattened leaves has been intensively studied. Classic microsurgery studies in potato and tomato suggest that the shoot apical meristem (SAM) communicates with the leaf primordia to promote leaf blade formation. More recently, it was found that polar auxin transport (PAT) could mediate this communication. However, it is unclear how the expression of leaf patterning genes is tailored by PAT routes originating from SAM. By combining experimental observations and computer model simulations, we show that microsurgical incisions and local inhibition of PAT in tomato interfere with auxin transport toward the leaf margins, reducing auxin response levels and altering the leaf blade shape. Importantly, oval auxin responses result in the bipolar expression of SlLAM1 that determines leaf blade formation. Furthermore, wounding caused by incisions promotes degradation of SlREV, a known regulator of leaf polarity. Additionally, computer simulations suggest that local auxin biosynthesis in early leaf primordia could remove necessity for external auxin supply originating from SAM, potentially explaining differences between species. Together, our findings establish how PAT near emerging leaf primordia determines spatial auxin patterning and refines SlLAM1 expression in the leaf margins to guide leaf flattening.

Characterization and expression profiling of PIN auxin efflux transporters reveal their role in developmental and abiotic stress conditions in rice.

The auxin efflux transporter proteins called PINs ferry auxin from its source to sinks in particular directions depending on their polar localizations in the plasma membrane, thus facilitating the development of the entire plant architecture. The rice genome has 12 PIN genes distributed over eight chromosomes. To study their roles in plant development, abiotic stress responsiveness, and shaping an auxin-dependent root architecture, a genome-wide analysis was carried out. Based on phylogeny, cellular localization, and hydrophilic loop domain size, the PINs were categorized into canonical and noncanonical PINs. PINs were found expressed in all of the organs of plants that emphasized their indispensable role throughout the plant's life cycle. We discovered that PIN5C and PIN9 were upregulated during salt and drought stress. We also found that regardless of its cellular level, auxin functioned as a molecular switch to turn on auxin biosynthesis genes. On the contrary, although PIN expression was upregulated upon initial treatment with auxin, prolonged auxin treatment not only led to their downregulation but also led to the development of auxin-dependent altered root formation in rice. Our study paves the way for developing stress-tolerant rice and plants with a desirable root architecture by genetic engineering.

Root architecture plasticity in response to endoparasitic cyst nematodes is mediated by damage signaling.

Plant root architecture plasticity in response to biotic stresses has not been thoroughly investigated. Infection by endoparasitic cyst nematodes induces root architectural changes that involve the formation of secondary roots at infection sites. However, the molecular mechanisms regulating secondary root formation in response to cyst nematode infection remain largely unknown. We first assessed whether secondary roots form in a nematode density-dependent manner by challenging wild-type Arabidopsis plants with increasing numbers of cyst nematodes (Heterodera schachtii). Next, using jasmonate-related reporter lines and knockout mutants, we tested whether tissue damage by nematodes triggers jasmonate-dependent secondary root formation. Finally, we verified whether damage-induced secondary root formation depends on local auxin biosynthesis at nematode infection sites. Intracellular host invasion by H. schachtii triggers a transient local increase in jasmonates, which activates the expression of ERF109 in a COI1-dependent manner. Knockout mutations in COI1 and ERF109 disrupt the nematode density-dependent increase in secondary roots observed in wild-type plants. Furthermore, ERF109 regulates secondary root formation upon H. schachtii infection via local auxin biosynthesis. Host invasion by H. schachtii triggers secondary root formation via the damage-induced jasmonate-dependent ERF109 pathway. This points at a novel mechanism underlying plant root plasticity in response to biotic stress.

Transcriptome Dynamics of Rooting Zone and Leaves during In Vitro Adventitious Root Formation in Eucalyptus nitens.

Wood properties and agronomic traits associated with fast growth and frost tolerance make Eucalyptus nitens a valuable forest alternative. However, the rapid age-related decline in the adventitious root (AR) formation (herein, meaning induction, initiation, and expression stages) limits its propagation. We analyzed transcriptomic profile variation in leaves and stem bases during AR induction of microcuttings to elucidate the molecular mechanisms involved in AR formation. In addition, we quantified expressions of candidate genes associated with recalcitrance. We delimited the ontogenic phases of root formation using histological techniques and Scarecrow and Short-Root expression quantification for RNA sequencing sample collection. We quantified the gene expressions associated with root meristem formation, auxin biosynthesis, perception, signaling, conjugation, and cytokinin signaling in shoots harvested from 2- to 36-month-old plants. After IBA treatment, 702 transcripts changed their expressions. Several were involved in hormone homeostasis and the signaling pathways that determine cell dedifferentiation, leading to root meristem formation. In part, the age-related decline in the rooting capacity is attributable to the increase in the ARR1 gene expression, which negatively affects auxin homeostasis. The analysis of the transcriptomic variation in the leaves and rooting zones provided profuse information: (1) To elucidate the auxin metabolism; (2) to understand the hormonal and signaling processes involved; (3) to collect data associated with their recalcitrance.

Endogenous auxin maintains embryonic cell identity and promotes somatic embryo development in Arabidopsis.

Somatic embryogenesis (SE), or embryo development from invitro cultured vegetative explants, can be induced in Arabidopsis by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Here, we explored the role of endogenous auxin [indole-3-acetic acid (IAA)] during 2,4-D and AHL15-induced SE. Using the pWOX2:NLS-YFP reporter, we identified three distinct developmental stages for 2,4-D and AHL15-induced SE in Arabidopsis, with these being (i) acquisition of embryo identity; (ii) formation of pro-embryos; and (iii) somatic embryo patterning and development. The acquisition of embryo identity coincided with enhanced expression of the indole-3-pyruvic acid auxin biosynthesis YUCCA genes, resulting in an enhanced pDR5:GFP-reported auxin response in the embryo-forming tissues. Chemical inhibition of the indole-3-pyruvic acid pathway did not affect the acquisition of embryo identity, but significantly reduced or completely inhibited the formation of pro-embryos. Co-application of IAA with auxin biosynthesis inhibitors in the AHL15-induced SE system rescued differentiated somatic embryo formation, confirming that increased IAA levels are important during the last two stages of SE. Our analyses also showed that polar auxin transport, with AUXIN/LIKE-AUX influx and PIN-FORMED1 efflux carriers as important drivers, is required for the transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that endogenous IAA biosynthesis and its polar transport are not required for the acquisition of embryo identity, but rather to maintain embryonic cell identity and for the formation of multicellular proembryos and their development into histodifferentiated embryos.

Auxin biosynthesis by Microbacterium testaceum Y411 associated with orchid aerial roots and their efficacy in micropropagation.

Root-associated bacteria strongly affect plant growth and development by synthesizing growth regulators and stress-relieving metabolites. The present study is mainly focused on assessing aerial root-associated bacteria of Rhynchostylis retusa (L.) Blume is an endemic epiphytic orchid responsible for auxin production and influencing plant growth. A bacterial isolate, Microbacterium testaceum Y411, was found to be the most active producer of indole-3-acetic acid (IAA). The maximum IAA production (170µg/mL) was recorded with the bacterium at optimum process parameters such as pH 7, temperature 30°C, and tryptophan 1000 µg/mL in a culture medium for 48h. The extracted auxin was purified and analyzed by FT-IR, HPLC, and HR-MS, indicating bacterial auxin has a similar mass value to 4-chloroindole-3-acetic acid auxin. Furthermore, the bacterial auxin was tested on in vitro propagation of orchid, Cymbidium aloifolium, and 90% seed germination was recorded in Murashige and Skoog's medium supplemented with bacterial auxin. The novel results obtained in this study are used for agricultural applications and the Microbacterium testaceum Y411 is a valuable biotechnological resource for a natural auxin.

Ways of Auxin Biosynthesis in Microorganisms

Among plant hormones, auxins, in particular indole-3-acetic acid (IAA), are the most studied and researched. Almost all groups of soil microorganisms, both plant-associated and non-plant-associated bacteria, fungi, and phytopathogenic microorganisms are capable of producing auxins. The development of preparations for crop production is directly related to the production of bacterial strains with high auxin-synthesizing potential, which is possible only with a full understanding of the ways of regulation and synthesis of auxins in bacteria. The synthesis of auxins in microorganisms can take place in two ways: by the gradual conversion of tryptophan to IAA (tryptophan-dependent pathway) or by the use of other intermediates (tryptophan-independent pathway). The latter is poorly clarified, and in the literature available today, there is only a small amount of information on the functioning of this pathway in microorganisms. The review presents literature data on the ways of auxin biosynthesis in different groups of microorganisms, as well as approaches to the intensification of indole-3-acetic acid synthesis. The formation of IAA from tryptophan can be carried out in the following ways: through indole-3-pyruvate, through indole-3-acetamide, and through indole-3-acetonitrile. The vast majority of available publications are related to the assimilation of tryptophan through the formation of indole-3-pyruvate as this pathway is the most common among microorganisms. Thus, it functions in rhizospheric, symbiotic, endophytic, and free-living bacteria. The concentration of synthesized IAA among natural strains is in the range from 260 to 1130 μg/mL. Microorganisms in which the indole-3-acetamide pathway functions are characterized by lower auxin-synthesizing ability compared to those that assimilate tryptophan through indole-3-pyruvate. These include bacteria of the genera Streptomyces, Pseudomonas, and Bradyrhizobium and fungi of the genus Fusarium. The level of synthesis of IAA in such microorganisms is from 1.17×10−4 to 255.6 μg/mL. To date, only two strains that assimilate tryptophan via the indole-3-acetonitrile pathway and form up to 31.5 μg/mL IAA have been described in the available literature. To intensify the synthesis of indole-3-acetic acid, researchers use two main approaches: the first consists in introducing into the culture medium of exogenous precursors of biosynthesis (usually tryptophan, less often indole-3-pyruvate, indole-3-acetamide, and indole-3-acetonitrile); the second — in increasing the expression of the corresponding genes and creating recomindolebinant strains-supersynthetics of IAA. The largest number of publications is devoted to increasing the synthesis of IAA in the presence of biosynthesis precursors. Depending on the type of bacteria, the composition of the nutrient medium, and the amount of exogenously introduced precursor, the synthesis of the final product was increased by 1.2—27 times compared to that before the intensifi cation. Thus, in the presence of 11 g/L tryptophan, Enterobacter sp. DMKU-RP206 synthesized 5.56 g/L, while in a medium without the precursor, it yielded only 0.45 g/L IAA. Recombinant strains Corynebacterium glutamicum ATCC 13032 and Escherichia coli MG165 formed 7.1 and 7.3 g/L IAA, respectively, when tryptophan (10 g/L) was added to the culture medium. The level of auxin synthesis in microorganisms may be increased under stress conditions (temperature, pH, biotic and abiotic stress factors), but in this case, the IAA concentration does not exceed 100 mg/L, and therefore this method of intensification cannot compete with the others above.

Tryptophan synthase ß subunit 1 affects stomatal phenotypes in Arabidopsis thaliana.

Stomata open in response to several environmental stimuli, such as light and low CO2. Plasma membrane (PM) H+-ATPase in guard cells plays a pivotal role for light-induced stomatal opening. In contrast, stomata close in response to the dark or plant hormone abscisic acid (ABA). However, molecular mechanisms of stomatal movements remain unclear. To elucidate the molecular mechanism of stomatal movements, we performed a genetic screen based on stomatal aperture-dependent weight decrease of detached leaves from EMS-treated Arabidopsis thaliana and isolated a rapid transpiration in detached leaves 2 (rtl2). The rtl2 mutant showed constitutive open-stomata phenotype with lower leaf temperature. ABA had no effect on stomatal aperture in rtl2. The rtl2 mutant also showed increased stomatal density, severe dwarf phenotype with pale green leaves and dark veins. Map-based analysis of the RTL2 locus revealed that the rtl2 mutant possesses a single nucleotide substitution, which induces amino acid substitution Gly162 to Glu in the tryptophan synthase ß subunit 1 (TSB1). The TSB1 encodes an enzyme in tryptophan (Trp) biosynthetic pathway. Amount of TSB1 protein was drastically reduced in rtl2 mutant. A different allele of tsb1 mutant (tsb1-1) also showed constitutive open-stomata phenotype with reduced TSB1 protein as in rtl2. Analyses of test-crossed plants of rtl2 and tsb1-1 showed open-stomata and dwarf phenotypes. These results indicate that a responsible gene for rtl2 is TSB1. We further investigated stomatal phenotype in mutants from Trp biosynthetic pathway, such as wei2-1 wei7-1, trp3-1, and tsb2-1. The trp3-1 mutant showed significant wider stomatal aperture as well as tsb1-1. Trp biosynthetic pathway closely relates to auxin biosynthesis. Then, we investigated auxin responsible genes and found that an expression of AUR3 was up in rtl2. In contrast, auxin had no effect on stomatal aperture in Arabidopsis and the phosphorylation status of PM H+-ATPase in guard cell protoplasts from Vicia faba. In addition, auxin antagonist had no effect on stomatal aperture. Interestingly, tsb1-1 grown under hydroponic culture system showed normal stomatal aperture by exogenously application of Trp. These results suggest that open stomata phenotype in tsb1-1 is due to Trp deficiency but not auxin.

Transcriptome and metabolome analyses reveal the efficiency of in vitro regeneration by TDZ pretreatment in mulberry

Thidiazuron (TDZ) is a plant growth regulator that is widely used as a defoliant and cytokinin-like compound. Additionally, mulberry is an economically important crop. However, regeneration is difficult in most mulberry species, and the regulatory mechanism controlling the regeneration process remains unclear. In this work, an efficient in vitro shoot regeneration system for mulberry was established by TDZ application. The results showed that the optimum adventitious bud induction, 70.83% in tetraploid M. notabilis cv. Chuanbei1 (CB1) and 96.82% in M. abla cv. Yunhua (YH), was obtained by the shoot pretreatment method using petiolate leaves as explants. The high-frequency regeneration petiolate leaf explants of YH treated with TDZ (T8) and the control (CK) were subjected to multiomic analyses. The comparison of the TDZ-treated with the CK groups identified 494 upregulated differentially expressed genes (DEGs) and 293 downregulated DEGs. GO analysis indicated that these DEGs were related mostly to integral component of membrane, transcription factor activity, and phosphorelay signal transduction system. Metabolomic analysis identified 1,889 compounds, including 205 differentially accumulated metabolites (DAMs). Of these DAMs, the number of lipids and lipid-like molecules was highest and accounted for 27.8% of all DAMs. Transcriptome profile, metabolomic, and phytohormone integrated analyses revealed that TDZ rearranged adenine-type cytokinins (CTKs) and negatively regulated endogenous CTK signal transduction. Moreover, TDZ treatment enhanced auxin signal transduction by increasing the expression of auxin flux carrier genes and downstream regulated genes without changing auxin biosynthesis in mulberry. In summary, this study provides a new method for mulberry in vitro regeneration and contributes to a better understanding of the transcriptional and metabolic changes that occur in mulberry after TDZ treatment.

Local auxin synthesis mediated by YUCCA4 induced during root-knot nematode infection positively regulates gall growth and nematode development.

Parasites and pathogens are known to manipulate the host's endogenous signaling pathways to facilitate the infection process. In particular, plant-parasitic root-knot nematodes (RKN) are known to elicit auxin response at the infection sites, to aid the development of root galls as feeding sites for the parasites. Here we describe the role of local auxin synthesis induced during RKN infection. Exogenous application of auxin synthesis inhibitors decreased RKN gall formation rates, gall size and auxin response in galls, while auxin and auxin analogues produced the opposite effects, re-enforcing the notion that auxin positively regulates RKN gall formation. Among the auxin biosynthesis enzymes, YUCCA4 (YUC4) was found to be dramatically up-regulated during RKN infection, suggesting it may be a major contributor to the auxin accumulation during gall formation. However, yuc4-1 showed only very transient decrease in gall auxin levels and did not show significant changes in RKN infection rates, implying the loss of YUC4 is likely compensated by other auxin sources. Nevertheless, yuc4-1 plants produced significantly smaller galls with fewer mature females and egg masses, confirming that auxin synthesized by YUC4 is required for proper gall formation and RKN development within. Interestingly, YUC4 promoter was also activated during cyst nematode infection. These lines of evidence imply auxin biosynthesis from multiple sources, one of them being YUC4, is induced upon plant endoparasitic nematode invasion and likely contribute to their infections. The coordination of these different auxins adds another layer of complexity of hormonal regulations during plant parasitic nematode interaction.

Identification of the Karyopherin Superfamily in Maize and Its Functional Cues in Plant Development.

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin β (IMPβ) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPβ genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network.