Global gene expression profile and functional analysis reveal the conservation of reproduction-associated gene networks in Gossypium hirsutum.

KEY MESSAGE: Lastly, the bZIP gene family encompasses genes that have been reported to play a role in flower development, such as bZIP14 (FD). Notably, bZIP14 is essential for Flowering Locus T (FT) initiation of floral development in Arabidopsis (Abe et al. 2005). Cotton (Gossypium hirsutum L.) is the world's most extensively cultivated fiber crop. However, its reproductive development is poorly characterized at the molecular level. Thus, this study presents a detailed transcriptomic analysis of G. hirsutum at three different reproductive stages. We provide evidence that more than 64,000 genes are active in G. hirsutum during flower development, among which 94.33% have been assigned to functional terms and specific pathways. Gene set enrichment analysis (GSEA) revealed that the biological process categories of floral organ development, pollen exine formation, and stamen development were enriched among the genes expressed during the floral development of G. hirsutum. Furthermore, we identified putative Arabidopsis homologs involved in the G. hirsutum gene regulatory network (GRN) of pollen and flower development, including transcription factors such as WUSCHEL (WUS), INNER NO OUTER (INO), AGAMOUS-LIKE 66 (AGL66), SPOROCYTELESS/NOZZLE (SPL/NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), ABORTED MICROSPORES (AMS), and ASH1-RELATED 3 (ASHR3), which are known crucial genes for plant reproductive success. The cotton MADS-box protein-protein interaction pattern resembles the previously described patterns for AGAMOUS (AG), SEEDSTICK (STK), SHATTERPROOF (SHP), and SEPALLATA3 (SEP3) homolog proteins from Arabidopsis. In addition to serving as a resource for comparative flower development studies, this work highlights the changes in gene expression profiles and molecular networks underlying stages that are valuable for cotton breeding improvement.

Elevated CO2 increases biomass of Sorghum bicolor green prop roots under drought conditions via soluble sugar accumulation and photosynthetic activity.

Elevated [CO2 ] (E[CO2 ]) mitigates agricultural losses of C4 plants under drought. Although several studies have described the molecular responses of the C4 plant species Sorghum bicolor during drought exposure, few have reported the combined effects of drought and E[CO2 ] (E[CO2 ]/D) on the roots. A previous study showed that, among plant organs, green prop roots (GPRs) under E[CO2 ]/D presented the second highest increase in biomass after leaves compared with ambient [CO2 ]/D. GPRs are photosynthetically active and sensitive to drought. To understand which mechanisms are involved in the increase in biomass of GPRs, we performed transcriptome analyses of GPRs under E[CO2 ]/D. Whole-transcriptome analysis revealed several pathways altered under E[CO2 ]/D, among which photosynthesis was strongly affected. We also used previous metabolome data to support our transcriptome data. Activities associated with photosynthesis and central metabolism increased, as seen by the upregulation of photosynthesis-related genes, a rise in glucose and polyol contents, and increased contents of chlorophyll a and carotenoids. Protein-protein interaction networks revealed that proliferation, biogenesis, and homeostasis categories were enriched and contained mainly upregulated genes. The findings suggest that the previously reported increase in GPR biomass of plants grown under E[CO2 ]/D is mainly attributed to glucose and polyol accumulation, as well as photosynthesis activity and carbon provided by respiratory CO2 refixation. Our findings reveal that an intriguing and complex metabolic process occurs in GPRs under E[CO2 ]/D, showing the crucial role of these organs in plant drought /tolerance.

An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

MAIN CONCLUSION: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Discovery and functional characterization of novel cotton promoters with potential application to pest control.

KEY MESSAGE: pGhERF105 and pGhNc-HARBI1 promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficiently drive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derived nuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96 h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

Evaluation of the molecular and physiological response to dehydration of two accessions of the model plant Setaria viridis.

Water deficits are responsible for countless agricultural losses. Among the affected crops, C4 plants are of special interest due to their high water and nitrogen use efficiency. Two accessions of Setaria viridis (Ast-1 and A10.1) with contrasting responses to water deficit were used in the current work to better understand the mechanisms behind drought tolerance in C4 species. Our results showed that although the A10.1 accession exhibited a reduced size and lower Rfd values in comparison to Ast-1, it had overall higher Fv/Fm and lower NPQ values in well-watered conditions. The water deficit induction was performed with PEG-8000 at the grain-filling stage using dehydration cycles. Analysis of physiological measurements showed the A10.1 accession as being more tolerant to multiple water deficit exposures. In addition, PCA identified a clear difference in the pattern of drought response of the accessions. Four drought marker genes previously described in the literature were chosen to evaluate the response at the molecular level: SvP5CS2, SvDHN1, SvNAC6, and SvWRKY1. Besides confirming that Ast-1 is a more sensitive accession, the expression analysis revealed that SvNAC1 might better monitor drought stress, while SvWRKY1 was able to differentiate the two accessions. Distinct evolutionary histories of each accession may be behind their differences in response to water deficits.

Overexpression of the CaHB12 transcription factor in cotton (Gossypium hirsutum) improves drought tolerance.

The Coffea arabica HB12 gene (CaHB12), which encodes a transcription factor belonging to the HD-Zip I subfamily, is upregulated under drought, and its constitutive overexpression (35S:CaHB12OX) improves the Arabidopsis thaliana tolerance to drought and salinity stresses. Herein, we generated transgenic cotton events constitutively overexpressing the CaHB12 gene, characterized these events based on their increased tolerance to water deficit, and exploited the gene expression level from the CaHB12 network. The segregating events Ev8.29.1, Ev8.90.1, and Ev23.36.1 showed higher photosynthetic yield and higher water use efficiency under severe water deficit and permanent wilting point conditions compared to wild-type plants. Under well-irrigated conditions, these three promising transformed events showed an equivalent level of Abscisic acid (ABA) and decreased Indole-3-acetic acid (IAA) accumulation, and a higher putrescine/(spermidine + spermine) ratio in leaf tissues was found in the progenies of at least two transgenic cotton events compared to non-transgenic plants. In addition, genes that are considered as modulated in the A. thaliana 35S:CaHB12OX line were also shown to be modulated in several transgenic cotton events maintained under field capacity conditions. The upregulation of GhPP2C and GhSnRK2 in transgenic cotton events maintained under permanent wilting point conditions suggested that CaHB12 might act enhancing the ABA-dependent pathway. All these data confirmed that CaHB12 overexpression improved the tolerance to water deficit, and the transcriptional modulation of genes related to the ABA signaling pathway or downstream genes might enhance the defense responses to drought. The observed decrease in IAA levels indicates that CaHB12 overexpression can prevent leaf abscission in plants under or after stress. Thus, our findings provide new insights on CaHB12 gene and identify several promising cotton events for conducting field trials on water deficit tolerance and agronomic performance.

ABAP1 Plays a Role in the Differentiation of Male and Female Gametes in Arabidopsis thaliana.

The correct development of a diploid sporophyte body and a haploid gametophyte relies on a strict coordination between cell divisions in space and time. During plant reproduction, these divisions have to be temporally and spatially coordinated with cell differentiation processes, to ensure a successful fertilization. Armadillo BTB Arabidopsis protein 1 (ABAP1) is a plant exclusive protein that has been previously reported to control proliferative cell divisions during leaf growth in Arabidopsis. Here, we show that ABAP1 binds to different transcription factors that regulate male and female gametophyte differentiation, repressing their target genes expression. During male gametogenesis, the ABAP1-TCP16 complex represses CDT1b transcription, and consequently regulates microspore first asymmetric mitosis. In the female gametogenesis, the ABAP1-ADAP complex represses EDA24-like transcription, regulating polar nuclei fusion to form the central cell. Therefore, besides its function during vegetative development, this work shows that ABAP1 is also involved in differentiation processes during plant reproduction, by having a dual role in regulating both the first asymmetric cell division of male gametophyte and the cell differentiation (or cell fusion) of female gametophyte.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

MAIN CONCLUSION: Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

Insights Into Genetic and Molecular Elements for Transgenic Crop Development.

Climate change and the exploration of new areas of cultivation have impacted the yields of several economically important crops worldwide. Both conventional plant breeding based on planned crosses between parents with specific traits and genetic engineering to develop new biotechnological tools (NBTs) have allowed the development of elite cultivars with new features of agronomic interest. The use of these NBTs in the search for agricultural solutions has gained prominence in recent years due to their rapid generation of elite cultivars that meet the needs of crop producers, and the efficiency of these NBTs is closely related to the optimization or best use of their elements. Currently, several genetic engineering techniques are used in synthetic biotechnology to successfully improve desirable traits or remove undesirable traits in crops. However, the features, drawbacks, and advantages of each technique are still not well understood, and thus, these methods have not been fully exploited. Here, we provide a brief overview of the plant genetic engineering platforms that have been used for proof of concept and agronomic trait improvement, review the major elements and processes of synthetic biotechnology, and, finally, present the major NBTs used to improve agronomic traits in socioeconomically important crops.

Insights obtained using different modules of the cotton uceA1.7 promoter.

MAIN CONCLUSION: The structure of the cotton uceA1.7 promoter and its modules was analyzed; the potential of their key sequences has been confirmed in different tissues, proving to be a good candidate for the development of new biotechnological tools. Transcriptional promoters are among the primary genetic engineering elements used to control genes of interest (GOIs) associated with agronomic traits. Cotton uceA1.7 was previously characterized as a constitutive promoter with activity higher than that of the constitutive promoter from the Cauliflower mosaic virus (CaMV) 35S gene in various plant tissues. In this study, we generated Arabidopsis thaliana homozygous events stably overexpressing the gfp reporter gene driven by different modules of the uceA1.7 promoter. The expression level of the reporter gene in different plant tissues and the transcriptional stability of these modules was determined compared to its full-length promoter and the 35S promoter. The full-length uceA1.7 promoter exhibited higher activity in different plant tissues compared to the 35S promoter. Two modules of the promoter produced a low and unstable transcription level compared to the other promoters. The other two modules rich in cis-regulatory elements showed similar activity levels to full-length uceA1.7 and 35S promoters but were less stable. This result suggests the location of a minimal portion of the promoter that is required to initiate transcription properly (the core promoter). Additionally, the full-length uceA1.7 promoter containing the 5'-untranslated region (UTR) is essential for higher transcriptional stability in various plant tissues. These findings confirm the potential use of the full-length uceA1.7 promoter for the development of new biotechnological tools (NBTs) to achieve higher expression levels of GOIs in, for example, the root or flower bud for the efficient control of phytonematodes and pest-insects, respectively, in important crops.

Artemisia annua L. and photoresponse: from artemisinin accumulation, volatile profile and anatomical modifications to gene expression.

KEY MESSAGE: Blue and yellow light affected metabolism and the morphology. Blue and red promote the DOXP/MEP pathway. ADS gene expression was increased in plants cultivated under blue, promoting artemisinin content. Artemisinin-based combination therapies are the most effective treatment for highly lethal malaria. Artemisinin is produced in small quantities in the glandular trichomes of Artemisia annua L. Our aim was to evaluate the effect of light quality in A. annua cultivated in vitro under different light qualities, considering anatomical and morphological changes, the volatile composition, artemisinin content and the expression of two key enzymes for artemisinin biosynthesis. Yellow light is related to the increase in the number of glandular trichomes and this seemed to positively affect the molecular diversity in A. annua. Yellow light-stimulated glandular trichome frequency without triggered area enhancement, whereas blue light stimulated both parameters. Blue light enhanced the thickness of the leaf epidermis. The B-promoting effect was due to increased cell size and not to increased cell numbers. Green and yellow light positively influenced the volatile diversity in the plantlets. Nevertheless, blue and red light seemed to promote the DOXP/MEP pathway, while red light stimulates MVA pathway. Amorpha-4,11-diene synthase gene expression was significantly increased in plants cultivated under blue light, and not red light, promoting artemisinin content. Our results showed that light quality, more specifically blue and yellow light, positively affected secondary metabolism and the morphology of plantlets. It seemed that steps prior to the last one in the artemisinin biosynthesis pathway could be strongly influenced by blue light. Our work provides an alternative method to increase the amount of artemisinin production in A. annua without the use of transgenic plants, by the employment of blue light.

Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity.

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here.

Anatomy and ultrastructure of embryonic leaves of the C4 species Setaria viridis.

Background and Aims: Setaria viridis is being promoted as a model C4 photosynthetic plant because it has a small genome (~515 Mb), a short life cycle (~60 d) and it can be transformed. Unlike other C4 grasses such as maize, however, there is very little information about how C4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves. Methods: Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy. Key Results: Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1-3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis. Conclusions: The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C4 grasses.

Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean.

Drought stress is the main limiting factor of soybean yield. Currently, genetic engineering has been one important tool in the development of drought-tolerant cultivars. A widely used strategy is the fusion of genes that confer tolerance under the control of the CaMV35S constitutive promoter; however, stress-responsive promoters would constitute the best alternative to the generation of drought-tolerant crops. We characterized the promoter of α-galactosidase soybean (GlymaGAL) gene that was previously identified as highly up-regulated by drought stress. The ß-glucuronidase (GUS) activity of Arabidopsis transgenic plants bearing 1000- and 2000-bp fragments of the GlymaGAL promoter fused to the uidA gene was evaluated under air-dried, polyethylene glycol (PEG) and salt stress treatments. After 24 h of air-dried and PEG treatments, the pGAL-2kb led to an increase in GUS expression in leaf and root samples when compared to the control samples. These results were corroborated by qPCR expression analysis of the uidA gene. The pGAL-1kb showed no difference in GUS activity between control and treated samples. The pGAL-2kb promoter was evaluated in transgenic soybean roots, leading to an increase in EGFP expression under air-dried treatment. Our data indicates that pGAL-2kb could be a useful tool in developing drought-tolerant cultivars by driving gene expression.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones.

KEY MESSAGE: Expression analysis of the AG -subfamily members from G. hirsutum during flower and fruit development. Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean

Abstract Drought stress is the main limiting factor of soybean yield. Currently, genetic engineering has been one important tool in the development of drought-tolerant cultivars. A widely used strategy is the fusion of genes that confer tolerance under the control of the CaMV35S constitutive promoter; however, stress-responsive promoters would constitute the best alternative to the generation of drought-tolerant crops. We characterized the promoter of α-galactosidase soybean (GlymaGAL) gene that was previously identified as highly up-regulated by drought stress. The β-glucuronidase (GUS) activity of Arabidopsis transgenic plants bearing 1000- and 2000-bp fragments of the GlymaGAL promoter fused to the uidA gene was evaluated under air-dried, polyethylene glycol (PEG) and salt stress treatments. After 24 h of air-dried and PEG treatments, the pGAL-2kb led to an increase in GUS expression in leaf and root samples when compared to the control samples. These results were corroborated by qPCR expression analysis of the uidA gene. The pGAL-1kb showed no difference in GUS activity between control and treated samples. The pGAL-2kb promoter was evaluated in transgenic soybean roots, leading to an increase in EGFP expression under air-dried treatment. Our data indicates that pGAL-2kb could be a useful tool in developing drought-tolerant cultivars by driving gene expression.

Selection of Reference Genes for Quantitative Real-Time RT-PCR Studies in Tomato Fruit of the Genotype MT-Rg1.

Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

Promoter isolation and characterization of GhAO-like1, a Gossypium hirsutum gene similar to multicopper oxidases that is highly expressed in reproductive organs.

Cotton is one of the most economically important cultivated crops. It is the major source of natural fiber for the textile industry and an important target for genetic modification for both biotic stress and herbicide tolerance. Therefore, the characterization of genes and regulatory regions that might be useful for genetic transformation is indispensable. The isolation and characterization of new regulatory regions is of great importance to drive transgene expression in genetically modified crops. One of the major drawbacks in cotton production is pest damage; therefore, the most promising, cost-effective, and sustainable method for pest control is the development of genetically resistant cotton lines. Considering this scenario, our group isolated and characterized the promoter region of a MCO (multicopper oxidase) from Gossypium hirsutum, named GhAO-like1 (ascorbate oxidase-like1). The quantitative expression, together with the in vivo characterization of the promoter region reveals that GhAO-like1 has a flower- and fruit-specific expression pattern. The GUS activity is mainly observed in stamens, as expected considering that the GhAO-like1 regulatory sequence is enriched in cis elements, which have been characterized as a target of reproductive tissue specific transcription factors. Both histological and quantitative analyses in Arabidopsis thaliana have confirmed flower (mainly in stamens) and fruit expression of GhAO-like1. In the present paper, we isolated and characterized both in silico and in vivo the promoter region of the GhAO-like1 gene. The regulatory region of GhAO-like1 might be useful to confer tissue-specific expression in genetically modified plants.