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.

Potassium supply promotes the mitigation of NaCl-induced effects on leaf photochemistry, metabolism and morphology of Setaria viridis.

Soil salinity has the potential to severely affect crop performance. To maintain cell functioning and improve salt tolerance, the maintenance of K+ homeostasis is crucial in several plant metabolism processes. Besides, potassium fertilization can efficiently alleviate the perilous effects of salinity. We characterized impacts in Setaria viridis exposed to NaCl and KCl to underlying photochemistry mechanisms, K+ and Na+ shoot contents, enzymatic activity, electrolytic leakage, and morphological responses focusing on non-stomatal limitation of photosynthesis. Plants were exposed to sodium chloride (NaCl; 0, 150 and 250 mM) and potassium chloride (KCl; 0, 5, 9 mM). The exposure to NaCl affected S. viridis leaves morphological and physiologically. Plants submitted to 150 mM showed reductions in performance indexes (PIabs and PItotal; JIP-test), and the presence of positive K- and L-bands. Plants exposed to 250 mM exhibited blockage in electron flow further than QA within 48h and permanent photoinhibition at 96 h. The presence of 9 and 5 mM of KCl counteracted the effects of NaCl on plants submitted to 150 mM, concomitant with increases in K+ accumulation and cell turgidity conservation, causing positive effects in plant growth and metabolism. Neither KCl concentrations were effective in reducing NaCl-induced effects on plants exposed to 250 mM of NaCl. Our results support the conclusion that greater availability of K+ alleviates the harmful effects of salinity in S. viridis under moderate stress and that application of KCl as means of lightning saline stress has a concentration and a salt level limit that must be experimentally determined.

A nonproteinaceous Fusarium cell wall extract triggers receptor-like protein-dependent immune responses in Arabidopsis and cotton.

Fusarium wilt caused by the ascomycete fungus Fusarium oxysporum is a devastating disease of many economically important crops. The mechanisms underlying plant responses to F. oxysporum infections remain largely unknown. We demonstrate here that a water-soluble, heat-resistant and nonproteinaceous F. oxysporum cell wall extract (FoCWE) component from multiple F. oxysporum isolates functions as a race-nonspecific elicitor, also termed pathogen-associated molecular pattern (PAMP). FoCWE triggers several demonstrated immune responses, including mitogen-activated protein (MAP) kinase phosphorylation, reactive oxygen species (ROS) burst, ethylene production, and stomatal closure, in cotton and Arabidopsis. Pretreated FoCWE protects cotton seeds against infections by virulent F. oxysporum f. sp. vasinfectum (Fov), and Arabidopsis plants against the virulent bacterium, Pseudomonas syringae, suggesting the potential application of FoCWEs in crop protection. Host-mediated responses to FoCWE do not appear to require LYKs/CERK1, BAK1 or SOBIR1, which are commonly involved in PAMP perception and/or signalling. However, FoCWE responses and Fusarium resistance in cotton partially require two receptor-like proteins, GhRLP20 and GhRLP31. Transcriptome analysis suggests that FoCWE preferentially activates cell wall-mediated defence, and Fov has evolved virulence mechanisms to suppress FoCWE-induced defence. These findings suggest that FoCWE is a classical PAMP that is potentially recognised by a novel pattern-recognition receptor to regulate cotton resistance to Fusarium infections.

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.

Identification of Players Controlling Meristem Arrest Downstream of the FRUITFULL-APETALA2 Pathway.

The end of the reproductive phase in monocarpic plants is determined by a coordinated arrest of all active meristems, a process known as global proliferative arrest (GPA). GPA is linked to the correlative control exerted by developing seeds and, possibly, the establishment of strong source-sink relationships. It has been proposed that the meristems that undergo arrest at the end of the reproductive phase behave at the transcriptomic level as dormant meristems, with low mitotic activity and high expression of abscisic acid response genes. Meristem arrest is also controlled genetically. In Arabidopsis (Arabidopsis thaliana), the MADS-box transcription factor FRUITFULL induces GPA by directly repressing genes of the APETALA2 (AP2) clade. The AP2 genes maintain shoot apical meristem (SAM) activity in part by keeping WUSCHEL expression active, but the mechanisms downstream of this pathway remain elusive. To identify target genes, we performed a transcriptomic analysis, inducing AP2 activity in meristems close to arrest. Our results suggest that AP2 controls meristem arrest by repressing genes related to axillary bud dormancy in the SAM and negative regulators of cytokinin signaling. In addition, our analysis indicates that genes involved in the response to environmental signals also respond to AP2, suggesting that it could modulate the end of flowering by controlling responses to both endogenous and exogenous signals. Our results support the previous observation that at the end of the reproductive phase the arrested SAM behaves as a dormant meristem, and they strongly support AP2 as a master regulator of this process.

Physiological and molecular responses of Setaria viridis to osmotic stress.

Drought-tolerant species, such as Setaria viridis, a C4 model plant, make physiological and biochemical adjustments water limitation and recover from the stress upon its release. We investigated S. viridis (A10.1 accession) responses to continuing osmotic stress. The osmotic stress was imposed using polyethylene glycol (PEG) 8000 (7.5%) for 10 days. Morphological traits and stomatal conductance were measured daily for the 10 days. On days 6 and 10, the following traits were measured separately for root and shoot: relative water content (RWC), osmotic potential (OP), electrolytic leakage (EL), and proline content. qPCR analysis was used to evaluate the expression of five selected genes in roots (SvLEA, SvDREB1C, SvPIP2-1, SvHSP20, and SvP5CS2), and chlorophyll a fluorescence was measured on three key days. The morphological data demonstrated a drastic reduction in shoot biomass as an effect of water deficit caused by the osmotic stress. Shoot biomass reduction could be associated with putative ABA-dependent signaling involved in SvDREB1C expression. Stomatal conductance and photosynthesis were severely affected up until day 6, however, stomatal conductance and some photosynthetic parameters such as FV/FM, ABS/RC, and DI0/RC showed total or slight recovery on day 10. Root EL decreased in treated plants suggesting an investment in membrane protection by osmoregulator expression such as dehydrin (SvLEA) and proline (SvP5CS2) genes. Our data suggest that S. viridis exhibited a partial recovery from an imposed and constant osmotic stress within 10 days.

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.

Short-term responses of soybean roots to individual and combinatorial effects of elevated [CO2] and water deficit.

Climate change increasingly threatens plant growth and productivity. Soybean (Glycine max) is one of the most important crops in the world. Although its responses to increased atmospheric carbon dioxide concentration ([CO2]) have been previously studied, root molecular responses to elevated [CO2] (E[CO2]) or the combination/interaction of E[CO2] and water deficit remain unexamined. In this study, we evaluated the individual and combinatory effects of E[CO2] and water deficit on the physiology and root molecular responses in soybean. Plants growing under E[CO2] exhibited increased photosynthesis that resulted in a higher biomass, plant height, and leaf area. E[CO2] decreased the transcripts levels of genes involved in iron uptake and transport, antioxidant activity, secondary metabolism and defense, and stress responses in roots. When plants grown under E[CO2] are treated with instantaneous water deficit, E[CO2] reverted the expression of water deficit-induced genes related to stress, defense, transport and nutrient deficiency. Furthermore, the interaction of both treatments uniquely affected the expression of genes. Both physiological and transcriptomic analyses demonstrated that E[CO2] may mitigate the negative effects of water deficit on the soybean roots. In addition, the identification of genes that are modulated by the interaction of E[CO2] and water deficit suggests an emergent response that is triggered only under this specific condition.

Characterization of Laguncularia racemosa transcriptome and molecular response to oil pollution.

Mangroves are ecosystems of economic and ecological importance. Laguncularia racemosa (Combretaceae), popularly known as white mangrove, is a species that greatly contributes to the community structure of neotropical and West African mangrove forests. Despite the significance of these ecosystems, they have been destroyed by oil spills that can cause yellowing of leaves, increased sensitivity to other stresses and death of trees. However, the molecular response of plants to oil stress is poorly known. In this work, Illumina reads were de novo assembled into 46,944 transcripts of L. racemosa roots and leaves, including putative isoform variants. In addition to improving the genomic information available for mangroves, the L. racemosa assembled transcriptome allowed us to identify reference genes to normalize quantitative real-time PCR (qPCR) expression data from oil-stressed mangrove plants, which were used in RNASeq validation. The analysis of expression changes induced by the oil exposure revealed 310 and 286 responsive transcripts of leaves and roots, respectively, mainly up-regulated. Enriched GO categories related to chloroplasts and photosynthesis were found among both leaf and root oil-responsive transcripts, while "response to heat" and "response to hypoxia" were exclusively enriched in leaves and roots, respectively. The comparison of L. racemosa 12-h-oil-stressed leaf expression profile to previous Arabidopsis heat-stress studies and co-expression evidence also pointed to similarities between the heat and oil responses, in which the HSP-coding genes seem to play a key role. A subset of the L. racemosa oil-responsive root genes exhibited similar up-regulation profiles to their Arabidopsis homologs involved in hypoxia responses, including the HRA1 and LBD41 TF-coding genes. Genes linked to the ethylene pathway such as those coding for ERF TFs were also modulated during the L. racemosa root response to oil stress. Taken together, these results show that oil contamination affects photosynthesis, protein metabolism, hypoxia response and the ethylene pathway in L. racemosa 12-h-oil-exposed leaves and roots.

Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum and Gossypium raimondii).

The MADS-box gene family encodes transcription factors that share a highly conserved domain known to bind to DNA. Members of this family control various processes of development in plants, from root formation to fruit ripening. In this work, a survey of diploid (Gossypium raimondii and Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton genomes found a total of 147, 133 and 207 MADS-box genes, respectively, distributed in the MIKC, Mα, Mß, Mγ, and Mδ subclades. A comparative phylogenetic analysis among cotton species, Arabidopsis, poplar and grapevine MADS-box homologous genes allowed us to evaluate the evolution of each MADS-box lineage in cotton plants and identify sequences within well-established subfamilies. Chromosomal localization and phylogenetic analysis revealed that G. raimondii and G. arboreum showed a conserved evolution of the MIKC subclade and a distinct pattern of duplication events in the Mα, Mγ and Mδ subclades. Additionally, G. hirsutum showed a combination of its parental subgenomes followed by a distinct evolutionary history including gene gain and loss in each subclade. qPCR analysis revealed the expression patterns of putative homologs in the AP1, AP3, AGL6, SEP4, AGL15, AG, AGL17, TM8, SVP, SOC and TT16 subfamilies of G. hirsutum. The identification of putative cotton orthologs is discussed in the light of evolution and gene expression data from other plants. This analysis of the MADS-box genes in Gossypium species opens an avenue to understanding the origin and evolution of each gene subfamily within diploid and polyploid species and paves the way for functional studies in cotton species.

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.