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Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

Low-temperature atmospheric pressure plasma has been used in various fields such as plasma medicine, agriculture, food safety and storage, and food manufacturing. In the field of plasma agriculture, plasma treatment improves seed germination, plant growth, and resistance to abiotic and biotic stresses, allows pesticide removal, and enhances biomass and yield. Currently, the complex molecular mechanisms of plasma treatment in plasma agriculture are fully unexplored, especially those related to seed germination and plant growth. Therefore, in this review, we have summarized the current progress in the application of the plasma treatment technique in plants, including plasma treatment methods, physical and chemical effects, and the molecular mechanism underlying the effects of low-temperature plasma treatment. Additionally, we have discussed the interactions between plasma and seed germination that occur through seed coat modification, reactive species, seed sterilization, heat, and UV radiation in correlation with molecular phenomena, including transcriptional and epigenetic regulation. This review aims to present the mechanisms underlying the effects of plasma treatment and to discuss the potential applications of plasma as a powerful tool, priming agent, elicitor or inducer, and disinfectant in the future.

Rice cultivation needs extensive amounts of water. Moreover, increased frequency of droughts and water scarcity has become a global concern for rice cultivation. Hence, optimization of water use is crucial for sustainable agriculture. Here, we characterized

Tobacco is an excellent model plant to study developmental processes and cellular biology. Surface dielectric barrier discharge (sDBD) was used to generate plasma activated water (PAW), and the PAW was indirectly applied to study tobacco plant growth during the early developmental stages. Solid media were treated with the PAW for 8- and 18-day-old seedlings, and the effects on both the vegetative and root phenotypes were observed during the early developmental stages. In the vegetative tissue, the cotyledon size was large for the seedling that was cultivated by plasma activated water for the 12 min PAW (PAW12) treatment. The PAW dramatically elongated the cotyledon by 40% due to increased cell expansion in the leaf-length direction, not cell proliferation. In addition to the vegetative tissue, the root length due to the PAW was significantly enhanced at the primary root with elongated root hairs. According to molecular analysis, the NT_ COBRA-LIKE 9 (COBL) and two NT_ XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) genes, NT_XTH9 and NT_XTH15, were significantly over-expressed by the PAW treatment compared with untreated deionized water (DW), indicating that the PAW effects are from modulating the expression of target genes. These results demonstrate that PAW might promote tobacco plant growth both in the cotyledon and root hair tissues via the NT_COBL, NT_XTH9 and NT_XTH15 genes.

In this study, genetic engineering methods, ranging from gene transformation to gene editing, were efficiently conducted on cotyledon explants in Solanum nigrum. Organogenic calli were observed on the explants following 10 days of growth on medium supplemented with 2 mg/L zeatin and 0.2 mg/L indole acetic acid (IAA), which we suggest to be an efficient shoot initiation medium (SM1). In vitro shoot production was enhanced in explants grown on media supplemented with 1 mg/L zeatin and 0.1 mg/L IAA, which we used as a proper shoot differentiation medium (SM2) in S. nigrum shoot regeneration. Direct infection of explants with Agrobacterium harboring CRISPR/Cas9 constructs can simplify the method of Agrobacterium-mediated T-DNA transformation by skipping the preincubation step. The method was applied to deliver transgenes for genome editing, such as CRISPR/Cas9 DNA. SnLazy1 locus, considered to be orthologs of tomato Lazy1, was edited using the CRISPR/Cas9 system in S. nigrum. Two independent deletion snlazy1-cr alleles were successively inherited and showed stem growth in a relatively downward direction. Our method offers rapid and simple procedure for gene transformation and genome editing that could be applicable for the enhancement of beneficial traits for precision plant breeding and engineering quantitative trait loci in S. nigrum.

Two novel bacterial strains, designated as BT186

Solanum nigrum, known as black nightshade, is a medicinal plant that contains many beneficial metabolites in its fruit. The molecular mechanisms underlying the synthesis of these metabolites remain uninvestigated due to limited genetic information. Here, we identified 47,470 unigenes of S. nigrum from three different tissues by de novo transcriptome assembly, and 78.4% of these genes were functionally annotated. Moreover, gene ontology (GO) analysis using 18,860 differentially expressed genes (DEGs) revealed tissue-specific gene expression regulation. We compared gene expression patterns between S. nigrum and tomato (S. lycopersicum) in three tissue types. The expression patterns of carotenoid biosynthetic genes were different between the two species. Comparison of the expression patterns of flavonoid biosynthetic genes showed that 9 out of 14 enzyme-coding genes were highly upregulated in the fruit of S. nigrum. Using CRISPR-Cas9-mediated gene editing, we knocked out the R2R3-MYB transcription factor SnAN2 gene, an ortholog of S. lycopersicum ANTHOCYANIN 2. The mutants showed yellow/green fruits, suggesting that SnAN2 plays a major role in anthocyanin synthesis in S. nigrum. This study revealed the connection between gene expression regulation and corresponding phenotypic differences through comparative analysis between two closely related species and provided genetic resources for S. nigrum.

A Gram-negative, short rod-shaped, and pink-pigmented bacterial strain, designated MA1

In plants, vegetative and reproductive development are associated with agronomically important traits that contribute to grain yield and biomass. Zinc finger homeodomain (ZF-HD) transcription factors (TFs) constitute a relatively small gene family that has been studied in several model plants, including Arabidopsis thaliana L. and Oryza sativa L. The ZF-HD family members play important roles in plant growth and development, but their contribution to the regulation of plant architecture remains largely unknown due to their functional redundancy. To understand the gene regulatory network controlled by ZF-HD TFs, we analyzed multiple loss-of-function mutants of ZF-HD TFs in Arabidopsis that exhibited morphological abnormalities in branching and flowering architecture. We found that ZF-HD TFs, especially HB34, negatively regulate the expression of miR157 and positively regulate SQUAMOSA PROMOTER BINDING–LIKE 10 (SPL10), a target of miR157. Genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis revealed that miR157D and SPL10 are direct targets of HB34, creating a feed-forward loop that constitutes a robust miRNA regulatory module. Network motif analysis contains overrepresented coherent type IV feedforward motifs in the amiR zf-HD and hbq mutant background. This finding indicates that miRNA-mediated ZF-HD feedforward modules modify branching and inflorescence architecture in Arabidopsis. Taken together, these findings reveal a guiding role of ZF-HD TFs in the regulatory network module and demonstrate its role in plant architecture in Arabidopsis.

The control of flowering time is a major contributing factor to the improvement of crop yield by optimizing plant growth in a crop cycle. Genetic variants that determine flowering time can provide insights into optimizing flowering time for higher yields and other beneficial traits in tomato crops. Here, we examined a collection of flowering time variants to assess their effects on biomass and total tomato yields. Five late flowering (lf), thirteen large plant (lp), and seven floral homeotic (fh) mutants were identified as flowering time variants that could be rearranged according to leaf production in the primary shoot meristem (PSM). A flowering time continuum of mutants was translated into a positive continuum of biomass yield with more leaves, branches, and floral organs. The flowering time continuum showed an optimal curve of fruit yield, indicating a certain late flowering time as optimal for fruit yield, with the yield gradually decreasing in both directions with earlier or later flowering times. We isolated lf1, lf10, lp22, and fh13 as high-yielding genotypes with optimal flowering time, showing a new balance between the vegetative and flowering phases of tomato. Additionally, lp8, fh8, and fh15 produced extremely high biomass in leaves, axillary shoots, and floral organs due to late flowering in shoot apices with additional production of floral organs and lateral shoot. Our new late-flowering variants provide new genetic resources that can be used to optimize crop yield by fine-tuning flowering time, and future molecular studies could be conducted by revisiting our yield model.

A Gram-stain-negative, aerobic, non-motile and yellow-colored bacterium, strain 17J57-3 T, was isolated from soil collected in Pyeongchang city, Korea. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 17J57-3 T formed a distinct lineage within the family Oxalobacteraceae (order Burkholderiales, class Betaproteobacteria). Strain 17J57-3 T was the most closely related to Noviherbaspirillum humi U15T (96.4% 16S rRNA gene sequence similarity) and Noviherbaspirillum massiliense JC206T (96.2%). The draft genome size of strain 17J57-3 T was 6,117,206 bp. Optimal growth occurred at 30 °C, pH 7.0 without NaCl. The predominant cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c) and C16:0. The major respiratory quinone was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Biochemical, chemotaxonomic and phylogenetic analyses indicated that strain 17J57-3 T represents a novel bacterial species within the genus Noviherbaspirillum, for which the name Noviherbaspirillum galbum is proposed. The type strain of Noviherbaspirillum galbum is 17J57-3 T (= KCTC 62213 T = NBRC 114384 T).

Grain number per panicle (GNP) is a major determinant of grain yield in cereals. However, the mechanisms that regulate GNP remain unclear. To address this issue, we isolate a series of sorghum [Sorghum bicolor (L.) Moench] multiseeded (msd) mutants that can double GNP by increasing panicle size and altering floral development so that all spikelets are fertile and set grain. Through bulk segregant analysis by next-generation sequencing, we identify MSD1 as a TCP (Teosinte branched/Cycloidea/PCF) transcription factor. Whole-genome expression profiling reveals that jasmonic acid (JA) biosynthetic enzymes are transiently activated in pedicellate spikelets. Young msd1 panicles have 50% less JA than wild-type (WT) panicles, and application of exogenous JA can rescue the msd1 phenotype. Our results reveal a new mechanism for increasing GNP, with the potential to boost grain yield, and provide insight into the regulation of plant inflorescence architecture and development., Inflorescence architecture affects crop grain yield. Here, the authors deploy whole-genome sequencing-based bulk segregant analysis to identify the causal gene of a sorghum multi-seeded (msd) mutant and suggest MSD1 regulating the fertility of the pedicellate spikelets through jasmonic acid pathway.

Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU. 1 more strongly than WT potentially perturbing differentiation via sequestration of PU. 1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema. Refereed/Peer-reviewed

As in other cereal crops, the panicles of sorghum (Sorghum bicolor (L.) Moench) comprise two types of floral spikelets (grass flowers). Only sessile spikelets (SSs) are capable of producing viable grains, whereas pedicellate spikelets (PSs) cease development after initiation and eventually abort. Consequently, grain number per panicle (GNP) is lower than the total number of flowers produced per panicle. The mechanism underlying this differential fertility is not well understood. To investigate this issue, we isolated a series of EMS-induced multiseeded (msd) mutants that result in full spikelet fertility, effectively doubling GNP. Previously, we showed that MSD1 is a TCP (Teosinte branched/Cycloidea/PCF) transcription factor that regulates jasmonic acid (JA) biosynthesis, and ultimately floral sex organ development. Here, we show that MSD2 encodes a lipoxygenase (LOX) that catalyzes the first committed step of JA biosynthesis. Further, we demonstrate that MSD1 binds to the promoters of MSD2 and other JA pathway genes. Together, these results show that a JA-induced module regulates sorghum panicle development and spikelet fertility. The findings advance our understanding of inflorescence development and could lead to new strategies for increasing GNP and grain yield in sorghum and other cereal crops.SignificanceThrough a single base pair mutation, grain number can be increased by ~200% in the globally important crop Sorghum bicolor. This mutation affects the expression of an enzyme, MSD2, that catalyzes the jasmonic acid pathway in developing floral meristems. The global gene expression profile in this enzymatic mutant is similar to that of a transcription factor mutant, msd1, indicating that disturbing any component of this regulatory module disrupts a positive feedback loop that occurs normally due to regular developmental perception of jasmonic acid. Additionally, the MSD1 transcription factor is able to regulate MSD2 in addition to other jasmonic acid pathway genes, suggesting that it is a primary transcriptional regulator of this hormone signaling pathway in floral meristems.

Most flowering plants have evolved to coordinate proper floral transition in shoot apical meristems at an appropriate time during development, referred to as “flowering time.” In domesticated crops, shoot life time has been manipulated to enhance productivity via synchronizing flowering time to the local environment, thereby creating a balance between vegetative and reproductive plant architecture during the year. Rice, a typical single transitional crop, has acquired balance between prolonged tiller growth and a relatively short reproductive phase on individual shoot, resulting in increasing yield. Crops with sympodial growth, such as tomato, which undergo multiple floral transitions on the main shoot, have been artificially selected for the determination of shoot growth by investigating the dosage sensitivity of florigen activation controlling flowering time on individual shoots. Here, we summarize recent genetic-molecular studies on tomato, which have been subject to genetic manipulation of flowering time in an effort to optimize seed productivity. We also review advanced genetic approaches as potential new tools for the enhancement of crop yield by manipulating flowering time via the dosage effect of florigen.

Gramene (http://www.gramene.org) is an online, open source, curated resource for plant comparative genomics and pathway analysis designed to support researchers working in plant genomics, breeding, evolutionary biology, system biology, and metabolic engineering. It exploits phylogenetic relationships to enrich the annotation of genomic data and provides tools to perform powerful comparative analyses across a wide spectrum of plant species. It consists of an integrated portal for querying, visualizing and analyzing data for 44 plant reference genomes, genetic variation data sets for 12 species, expression data for 16 species, curated rice pathways and orthology-based pathway projections for 66 plant species including various crops. Here we briefly describe the functions and uses of the Gramene database.

Shoot apical meristems are stem cell niches that balance proliferation with the incorporation of daughter cells into organ primordia. This balance is maintained by CLAVATA-WUSCHEL feedback signaling between the stem cells at the tip of the meristem and the underlying organizing center. Signals that provide feedback from organ primordia to control the stem cell niche in plants have also been hypothesized, but their identities are unknown. Here we report FASCIATED EAR3 (FEA3), a leucine-rich-repeat receptor that functions in stem cell control and responds to a CLAVATA3/ESR-related (CLE) peptide expressed in organ primordia. We modeled our results to propose a regulatory system that transmits signals from differentiating cells in organ primordia back to the stem cell niche and that appears to function broadly in the plant kingdom. Furthermore, we demonstrate an application of this new signaling feedback, by showing that weak alleles of fea3 enhance hybrid maize yield traits.

A high-throughput screening system for Citrus lines were established with higher sugar and acid contents using Fourier transform infrared (FT-IR) spectroscopy in combination with multivariate analysis. FT-IR spectra confirmed typical spectral differences between the frequency regions of 950-1100 cm(-1), 1300-1500 cm(-1), and 1500-1700 cm(-1). Principal component analysis (PCA) and subsequent partial least square-discriminant analysis (PLS-DA) were able to discriminate five Citrus lines into three separate clusters corresponding to their taxonomic relationships. The quantitative predictive modeling of sugar and acid contents from Citrus fruits was established using partial least square regression algorithms from FT-IR spectra. The regression coefficients (R(2)) between predicted values and estimated sugar and acid content values were 0.99. These results demonstrate that by using FT-IR spectra and applying quantitative prediction modeling to Citrus sugar and acid contents, excellent Citrus lines can be early detected with greater accuracy.

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo.

The LONGIFOLIA1 (LNG1) gene of Arabidopsis regulates leaf shape by polar cell elongation independent of ROTUNDAFOLIA3 (ROT3). To expand our knowledge on the function of this gens in plant systems, Arabidopsis LNG1 (AtLNG1) was introduced both sense and antisense orientation under the control of 35S CaMV promoter into tobacco plants that lack AtLNG1 homolog. Resulting transgenic tobacco plants were analyzed by their phenotype, anatomy and transcript levels. AtLNG1-overexpressing tobacco lines showed increase in the leaf petiole and leaf blade compared with wild type tobacco line. The overexpressors also showed elongated palisade cells as well as epidermal cells in the leaf length direction, but no increase in cell number. Ectopic expression of AtLNG1 in tobacco plants also increased the expression of cell wall modification-related genes, such as NT_XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (NT_XTH9), NT_XTH15 and NT_XTH33, indicating that these genes appear to be target of AtLNG1. As results of molecular and cellular examination, AtLNG1 seemed to have a conserved functional role in shaping leaf morphology in both Arabidopsis and tobacco.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functionsof GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema Refereed/Peer-reviewed

Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene–gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.

In this work, we genetically characterized the function of Arabidopsis thaliana, LONGIFOLIA (LNG1), LNG2, LNG3, LNG4, their contribution to regulate vegetative architecture in plant. We used molecular and biophysical approaches to elucidate a gene function that regulates vegetative architecture, as revealed by the leaf phenotype and later effects on flowering patterns in Arabidopsis loss-of-function mutants. As a result, LNG genes play an important role in polar cell elongation by turgor pressure controlling the activation of XTH17 and XTH24. Plant vegetative architecture is related to important traits that later influence the floral architecture involved in seed production. Leaf morphology is the primary key trait to compose plant vegetative architecture. However, molecular mechanism on leaf shape determination is not fully understood even in the model plant A. thaliana. We previously showed that LONGIFOLIA (LNG1) and LONGIFOLIA2 (LNG2) genes regulate leaf morphology by promoting longitudinal cell elongation in Arabidopsis. In this study, we further characterized two homologs of LNG1, LNG3, and LNG4, using genetic, biophysical, and molecular approaches. Single loss-of-function mutants, lng3 and lng4, do not show any phenotypic difference, but mutants of lng quadruple (lngq), and lng1/2/3 and lng1/2/4 triples, display reduced leaf length, compared to wild type. Using the paradermal analysis, we conclude that the reduced leaf size of lngq is due to decreased cell elongation in the direction of longitudinal leaf growth, and not decreased cell proliferation. This data indicate that LNG1/2/3/4 are functionally redundant, and are involved in polar cell elongation in Arabidopsis leaf. Using a biophysical approach, we show that the LNGs contribute to maintain high turgor pressure, thus regulating turgor pressure-dependent polar cell elongation. In addition, gene expression analysis showed that LNGs positively regulate the expression of the cell wall modifying enzyme encoded by a multi-gene family, xyloglucan endotransglucosylase/hydrolase (XTH). Taking all of these together, we propose that LNG related genes play an important role in polar cell elongation by changing turgor pressure and controlling the activation of XTH17 and XTH24.

Arabidopsis plants adapt to high ambient temperature by a suite of morphological changes including elongation of hypocotyls and petioles and leaf hyponastic growth. These morphological changes are collectively called thermomorphogenesis and are believed to increase leaf cooling capacity by enhancing transpiration efficiency, thereby increasing tolerance to heat stress. The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) has been identified as a major regulator of thermomorphogenic growth. Here, we show that PIF4 promotes the expression of two homologous genes LONGIFOLIA1 (LNG1) and LONGIFOLIA2 (LNG2) that have been reported to regulate leaf morphology. ChIP-Seq analyses and ChIP assays showed that PIF4 directly binds to the promoters of both LNG1 and LNG2. The expression of LNG1 and LNG2 is induced by high temperature in wild type plants. However, the high temperature activation of LNG1 and LNG2 is abolished in the pif4 mutant, indicating that PIF4 directly regulates LNG1 and LNG2 expression in response to high ambient temperatures. We further show that the activities of LNGs are required for thermomorphogenic growth. The expression of auxin biosynthetic and responsive genes is decreased in the lng quadruple mutant, implying that LNGs promote thermomorphogenic growth by activating the auxin pathway. Together, our results demonstrate that LNG1 and LNG2 are directly regulated by PIF4 and are new components for the regulation of thermomorphogenesis.

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies. Refereed/Peer-reviewed

With 6 figures Abstract The Ser/Thr protein kinase subfamily of the novel Arabidopsis protein kinase (NAK) family is unique to plants and is not found in animals or prokaryotes, suggesting that NAKs play specific signalling roles in the control of plant growth and development. However, little is known about the function of NAK-type protein kinases. Here, we used a transgenic tomato plant system to examine the biological functions of AFSK, a NAK-type protein kinase isolated from apple fruits. Gain-of-function transgenic tomato plants containing the AFSK gene were developed by Agrobacterium-mediated transformation and plant regeneration. The plants had normal flower phenotype with respect to their overall shapes and sizes. However, the plants displayed decreased self-fertilization and early flower drop at the floral abscission zone following petal loss. These results suggest that AFSK affects pollen development and promotes floral abscission.

To test the effect of endo-β-1,4-glucanase (EGase) in strawberry, we produced transgenic strawberry plants that contained sense and antisense cDNA encoding strawberry EGase under the CaMV 35S promoter (CaMV35S-P) and the strawberry fruit dominant ascorbate peroxidase (APX) promoter. Independent transgenic lines were generated and the firmness of the fruit was characterized after harvest. Interestingly, transgenic lines that harbored the cDNA antisense under the CaMV 35S promoter were not generated, but transgenic lines that contained sense EGase cDNA (FraCel1) under the CaMV 35S promoter and sense and antisense EGase cDNA under the APX promoter were successfully obtained using a tissue culture system. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the steady-state transcript levels of CaMV35S-P:FraCel1 sense transgenic lines were dramatically increased in fruit evaluated at all stages, as well as in the leaves. Moreover, real-time PCR analysis demonstrated that the steady-state transcript level of FraCel1 in independent transgenic lines that contained antisense FraCel1 under the APX promoter was reduced in fruit during the turning and red stages when compared with wild-type strawberries. These results were consistent with the firmness of strawberries that contained the antisense FraCel1 under the APX promoter, which was 19–36% greater in the turning stage and 22–25% greater in the red stage when compared to wild-type strawberries. Taken together, these findings indicate that fruit-specific down-regulation of the EGase gene using the APX promoter is an effective technique for increasing fruit firmness in strawberries.

To modulate the soluble sugar content of strawberry fruits, we generated transgenic plants that incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit (FagpS) under the control of the strawberry fruit-dominant ascorbate peroxidase (APX) promoter (cv. Anther). Several independent transgenic lines were obtained and grown in the greenhouse for analysis of agronomic traits. Most transgenic fruit did not show significant differences in weight and hardness compared to control fruit. However, the starch content in fruit was decreased to 27–47% and the total soluble sugar content was increased to 16–37% in transgenic plants (analyzed by the HPLC of sugar composition at four different stages of fruit development). The sugar contents of fruits in transgenic lines were particularly higher than control fruits at the red stage. The results were consistent with northern analysis, which showed that the levels of AGPase mRNA drastically were reduced in the red stage of fruits in all the transgenic plants. In other tissues of transgenic plants, the FagpS mRNA expression level was similar to control plants. Our studies indicate that fruit-specific down-regulation of the AGPase gene might be an effective strategy for increasing sugar and decreasing starch content in strawberry.

A negative selectable marker gene, codA, was successfully co-transformed with a GUS reporter gene to develop selectable marker gene-free transgenic plants. The pNC binary vector contained a T-DNA harboring the codA gene next to the nptII gene, while a second binary vector, pHG, contained a GUS reporter gene. Tobacco plants ( Nicotiana tabacum cv. Samsun NN) were co-transformed via the mixture method with Agrobacterium tumefaciens LBA4404 strains harboring pNC and pHG, respectively. Seeds harvested from the co-transformants were sown on germination media containing 5-fluorocytosine (5-FC). Analysis of the progeny by GUS staining and PCR amplification revealed that all of the 5-FC-resistant R(1) plants were codA free, and that the codA gene segregated independently of the GUS gene. Because codA-free seedlings developed normally on 5-FC-containing medium, we suggest that co-transformation with negatively selectable markers is a viable method for the production of easily distinguished, selectable marker gene-free transgenic plants.

Using cotyledonary explants excised from seedlings germinated in vitro, efficient plant regeneration via organogenesis was established for two winter squash (Cucurbita maxima Duch.) cultivars. To establish optimal conditions for adventitious shoot induction, a variety of explants were prepared from seedlings of different ages and these were cultured using media containing different concentrations of 6-benzylaminopurine (BA). For both cultivars, plant regeneration was optimal when the proximal parts of cotyledons from 4-day-old seedlings were cultured on induction medium composed of Murashige and Skoog (MS) medium with 1 mg/l BA. After 3 weeks of culture in induction medium, 82 and 92% of explants from the two cultivars regenerated shoots. Adventitious shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA and the elongated shoots were successfully rooted in MS medium without growth regulators for 2 weeks. Flow cytometric analysis revealed that most of the regenerated plants were diploid.