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In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transportrelated genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the inability to produce functional pollen. The CMS-associated protein Orf507 (reported as Orf456 in previous researches) was previously identified as a candidate gene for mediating male sterility in pepper. Here, we performed yeast two-hybrid analysis to screen for interacting proteins, and found that the ATP synthase 6 kDa subunit containing a mitochondrial signal peptide (MtATP6) specifically interacted with Orf507. In addition, the two proteins were found to be interacted in vivo using bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Further functional characterization of Orf507 revealed that the encoded protein is toxic to bacterial cells. Analysis of tissue-specific expression of ATP synthase 6 kDa showed that the transcription level was much lower in anthers of the CMS line than in their wild type counterparts. In CMS plants, ATP synthase activity and content were reduced by more than half compared to that of the normal plants. Taken together, it can be concluded that reduced ATP synthase activity and ATP content might have affected pollen development in CMS plants. Here, we hypothesize that Orf507 might cause MtATP6 to be nonfunctional by changing the latter's conformation or producing an inhibitor that prevents the normal functioning of MtATP6. Thus, further functional analysis of mitochondrial Orf507 will provide insights into the mechanisms underlying CMS in plants.

A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid (18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F₂ population from a cross between C. chinense ‘sy-2' and wild-type C. chinense ‘No. 3341' showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold 00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response.

Main conclusionUse of Ultra-low gossypol cottonseed event as a scion in a graft combination confirmed that roots are not a source of terpenoids in the aboveground parts of a cotton plant.Gossypol and related terpenoids, derived from the same basic biosynthetic pathway, are present in the numerous lysigenous glands in the aboveground parts of a cotton plant. Roots, with sparse presence of such glands, do produce significant amount of gossypol and a different set of terpenoids. These compounds serve a defensive function against various pests and pathogens. This investigation was undertaken to examine whether gossypol produced in the roots can replenish the gossypol content of the cottonseed-glands that are largely devoid of this terpenoid in a genetically engineered event. Graft unions between a scion derived from the RNAi-based, Ultra-low gossypol cottonseed (ULGCS) event, TAM66274, and a rootstock derived from wild-type parental genotype, Coker 312 (Coker), were compared with various other grafts that served as controls. The results showed that the seeds developing within the scion of test grafts (ULGCS/Coker) continued to maintain the ultra-low gossypol levels found in the TAM66274 seeds. Molecular analyses confirmed that while the key gene involved in gland development showed normal activity in the developing embryos in the scion, two genes encoding the enzymes involved in gossypol biosynthesis were suppressed. Thus, the gene expression data confirmed the results obtained from biochemical measurements and collectively demonstrated that roots are not a source of gossypol for the aboveground parts of the cotton plant. These findings, combined with the results from previous investigations, support the assertion that gossypol and related terpenoids are produced in a highly localized manner in various organs of the cotton plant and are retained therein.

Summary In seeds and other parts of cultivated, tetraploid cotton (Gossypium hirsutum L.), multicellular groups of cells lysigenously form dark glands containing toxic terpenoids such as gossypol that defend the plant against pests and pathogens. Using RNA‐seq analysis of embryos from near‐isogenic glanded (Gl2Gl2Gl3Gl3) versus glandless (gl2gl2gl3gl3) plants, we identified 33 genes that expressed exclusively or at higher levels in embryos just prior to gland formation in glanded plants. Virus‐induced gene silencing against three gene pairs led to significant reductions in the number of glands in the leaves, and significantly lower levels of gossypol and related terpenoids. These genes encode transcription factors and have been designated the ‘Cotton Gland Formation’ (CGF) genes. No sequence differences were found between glanded and glandless cotton for CGF1 and CGF2 gene pairs. The glandless cotton has a transposon insertion within the coding sequence of the GoPGF (synonym CGF3) gene of the A subgenome and extensive mutations in the promoter of D subgenome homeolog. Overexpression of GoPGF (synonym CGF3) led to a dramatic increase in gossypol and related terpenoids in cultured cells, whereas CRISPR/Cas9 knockout of GoPGF (synonym CGF3) genes resulted in glandless phenotype. Taken collectively, the results show that the GoPGF (synonym CGF3) gene plays a critical role in the formation of glands in the cotton plant. Seed‐specific silencing of CGF genes, either individually or in combination, could eliminate glands, thus gossypol, from the cottonseed to render it safe as food or feed for monogastrics.

Weeds, which have been the bane of agriculture since the beginning of civilization, are managed manually, mechanically, and, more recently, by chemicals. However, chemical control options are rapidly shrinking due to the recent rise in the number of herbicide-resistant weeds in crop fields, with few alternatives on the horizon. Therefore, there is an urgent need for alternative weed suppression systems to sustain crop productivity while reducing our dependence on herbicides and tillage. Such a development will also allay some of the negative perceptions associated with the use of herbicide-resistance genes and heavy dependence on herbicides. Transgenic plants expressing the bacterial phosphite dehydrogenase (ptxD) gene gain an ability to convert phosphite (Phi) into orthophosphate [Pi, the metabolizable form of phosphorus (P)]. Such plants allow for a selective fertilization scheme, based on Phi as the sole source of P for the crop, while offering an effective alternative for suppressing weed growth. Here, we show that, when P is supplied in the form of Phi, ptxD-expressing cotton (Gossypium hirsutum L.) plants outcompete, in both artificial substrates and natural soils from agricultural fields, three different monocot and dicot weed species intentionally introduced in the experiments, as well as weeds naturally present in the tested soils. Importantly, the ptxD/Phi system proved highly efficacious in inhibiting the growth of glyphosate-resistant Palmer amaranth. With over 250 weed species resistant to currently available herbicides, ptxD-transgenic plants fertilized with Phi could provide an effective alternative to suppressing the growth of these weeds while providing adequate nutrition to the crop.

Key message This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium -mediated transformation. Under the ptxD /Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar /phosphinothricin (PPT) selection was used. The event recovery rates for nptII /kanamycin and hpt /hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD /Phi system. Thus, ptxD /Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

Allele mining is a method used to find undiscovered natural variations or induced mutations in a plant, and has become increasingly important as more genomic information is available in plants. A high-throughput method is required to facilitate the identification of novel alleles in a large number of samples. In this paper we describe the application of a high-resolution melting (HRM) method to detect natural variations and ethyl methane sulfonate (EMS)-induced mutations in Capsicum. We have scanned single polymorphic mutations in the first exon of the eIF4E gene, wherein the mutations confer resistance to potyviruses. Sixteen allelic variations out of 248 germplasm collections were identified using HRM analysis, and one accession carrying an allelic variation (pvrHRM1 13 ) was confirmed to be resistant to the TEV-HAT strain. In addition, five single polymorphic mutations in the eIF4E gene were identified in an EMS-induced mutant population. These results demonstrate that HRM allows for the rapid identification of new allelic variants in both natural and artificial mutant populations.

In order to understand the molecular genetic mechanisms of rice ( O ryza sativa ) organ development, we studied the narrow leaf2 narrow leaf3 ( nal2 nal3 ; hereafter nal2/3 ) double mutant, which produces narrow‐curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow‐thin grains. We found that narrow‐curly leaves resulted mainly from reduced lateral‐axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow‐thin grains. Map‐based cloning revealed that NAL 2 and NAL 3 are paralogs that encode an identical O s WOX 3 A ( O s NS ) transcriptional activator, homologous to NARROW SHEATH 1 ( NS 1) and NS 2 in maize and PRESSED FLOWER in A rabidopsis. O s WOX 3 A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development‐associated genes were altered in nal2/3 , and auxin transport‐related genes were significantly changed, leading to pin mutant‐like phenotypes such as more tillers and fewer lateral roots. O s WOX 3 A is involved in organ development in rice, lateral‐axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.