HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Molecular Characterization of Native Bacillus thuringiensis Strains from Root Nodules with Toxicity Against the Fall Armyworm (FAW, Spodoptera frugiperda) and Brinjal Ash Weevil (Myllocerus subfasciatus).

The fall armyworm is an exotic pest which destroys a wide variety of crops Querywhereas the brinjal ash weevil is a serious pest of eggplant and other solanaceous vegetables. The goal of this research is to find a sustainable and ecologically friendly bio-control agent for managing FAW and brinjal ash weevils. Twelve natural Bacillus thuringiensis strains were isolated from cowpea root nodules, and the Gram-positive cells with characteristic Bt crystal structures were discovered using phase contrast and scanning electron microscopy. There were bipyramidal, cuboidal, rhombus, and spherical crystals. The Bt cry gene content was characterized by PCR analysis, which revealed the presence of cry1, cry1I, cry3, cry7, cry7,8, cry14, cry26, and cry55 genes. The identity of Bt was confirmed by cloning and sequencing the cry genes. In the nucleotide sequences, no pseudo genes or indels were found in cry sequences. SDS-PAGE examination indicated the presence of bands ranging in size from 13 to 130 kDa, with 50-60 kDa being the most common. When compared to the control, the new native Bt strains were lethal, with pathogenicity ranging from 93 to 100% against S. frugiperda larvae and M. subfasciatus adults. The studies revealed that the native strains with conserved regions of 16S rRNA genes were compared to NCBI database sequences and classified as native Bt strains with 99-100% similarity to known Bt strains. In conclusion, native Bt strains from cowpea root nodules were shown to have bio-insecticidal activity against fall armyworm larvae and brinjal ash weevil adults.

Correction to: Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut.

The original version of this article contains the following duplicate figures, namely blots showing expression data of clones.

Expression analysis of drought stress specific genes in Peanut (Arachis hypogaea , L.).

Improving drought tolerance through gene manipulation has been of importance for modern agriculture, which requires identification and validation of candidate genes. Prospecting candidate genes from drought adapted crop species is of immense significance. To identify candidate stress responsive genes from adapted crop, we carried out expression analysis of a few drought responsive ESTs from Arachis hypogaea L. (peanut). The expression patterns of nine AhDR (Arachis hypogea drought responsive) clones were analysed under drought. Quantitative reverse transcription PCR analysis revealed stress responsive nature of the selected genes. The clones AhDR 118 (putative cyclin T-like), AhDR185 (aldehyde reductase-like), AhDR193 (cholin kinase-like) and AhDR 76 (proline amino peptidase-like) showed more than five fold increase in expression. Highly upregulated genes analysed for expression pattern against salinity at seedling level indicated that these genes provide cross protection. This paper is the first report indicating the association of peanut genes cyclin T, proline amino peptidase and choline kinase to drought tolerance, and the possible roles of these genes are discussed.

MAPK-dependent JA and SA signalling in Nicotiana attenuata affects plant growth and fitness during competition with conspecifics.

BACKGROUND: Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA)-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs), salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK) and WIPK (irWIPK) benefits the growth and fitness of plants competiting with wild type (WT) plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3) levels. RESULTS: As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels resulted from increased nitrogen or CO2 assimilation rates, and found no evidence that greater intake of these fitness-limiting resources were responsible. CONCLUSIONS: Signaling mediated by WIPK, but not SIPK, is associated with large fitness costs in competing N. attenuata plants, demonstrating the contrasting roles that these two MAPKs play in regulating the plants' growth-defense balance. We discuss the role of SIPK as an important regulator of plant fitness, possibly by modulating SA-JA crosstalk as mediated through ethylene signaling.

Activation of drought tolerant traits in crops: endophytes as elicitors.

Drought challenges crop production worldwide. The issue is aggravated by frequent drought episodes and unpredictable rainfall patterns associated with global climate change. While the efforts to breed drought-resistant crop varieties are progressing, the need of the hour is immediate strategies to sustain the yields of existing ones. As per recent studies, stress adaptive traits can be activated using specific elicitors. Endophytes that inhabit host plants asymptomatically are natural elicitors/bio-stimulators capable of activating host gene expression, conferring several benefits to the hosts. This review discusses the scope of using trait-specific endophytes in activating drought adaptive traits in crop varieties.

Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut.

Peanut, found to be relatively drought tolerant crop, has been the choice of study to characterize the genes expressed under gradual water deficit stress. Nearly 700 genes were identified to be enriched in subtractive cDNA library from gradual process of drought stress adaptation. Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Fifty genes (25 regulators and 25 functional related genes) selected based on dot blot experiments were tested for their stress responsiveness using northern blot analysis and confirmed their nature of differential regulation under different field capacity of drought stress treatments. ESTs generated from this subtracted cDNA library offered a rich source of stress-related genes including signaling components. Additional 50% uncharacterized sequences are noteworthy. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. At present functional validation cannot be deemed in peanut, hence as a proof of concept seven orthologues of drought induced genes of peanut have been silenced in heterologous N. benthamiana system, using virus induced gene silencing method. These results point out the functional importance for HSP70 gene and key regulators such as Jumonji in drought stress response.

Aberrant imprinting may underlie evolution of parthenogenesis.

Genomic imprinting confers parent-of-origin-specific gene expression, thus non-equivalent and complementary function of parental genomes. As a consequence, genomic imprinting poses an epigenetic barrier to parthenogenesis in sexual organisms. We report aberrant imprinting in Boechera, a genus in which apomicts evolved from sexuals multiple times. Maternal activation of a MADS-box gene, a homolog of which is imprinted and paternally expressed in the sexual relative Arabidopsis, is accompanied by locus-specific DNA methylation changes in apomicts where parental imprinting seems to be relaxed.

GBF3 transcription factor imparts drought tolerance in Arabidopsis thaliana.

Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

VRS2 regulates hormone-mediated inflorescence patterning in barley.

Plant architecture has clear agronomic and economic implications for crops such as wheat and barley, as it is a critical factor for determining grain yield. Despite this, only limited molecular information is available about how grain-bearing inflorescences, called spikes, are formed and maintain their regular, distichous pattern. Here we elucidate the molecular and hormonal role of Six-rowed spike 2 (Vrs2), which encodes a SHORT INTERNODES (SHI) transcriptional regulator during barley inflorescence and shoot development. We show that Vrs2 is specifically involved in floral organ patterning and phase duration by maintaining hormonal homeostasis and gradients during normal spike development and similarly influences plant stature traits. Furthermore, we establish a link between the SHI protein family and sucrose metabolism during organ growth and development that may have implications for deeper molecular insights into inflorescence and plant architecture in crops.

The Genetic Basis of Composite Spike Form in Barley and 'Miracle-Wheat'.

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. 'Compositum-Barley' and tetraploid 'Miracle-Wheat' (T. turgidum convar. compositum (L.f.) Filat.) display noncanonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation 'Miracle-Wheat' produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in 'Compositum-Barley,' i.e., compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched head(t) (bh(t)) locus regulating spike branching in tetraploid 'Miracle-Wheat.' Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bh(t) locus in a collection of mutant and wild-type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of 'Miracle-Wheat.' mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative downstream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat's yield potential.

Contrapuntal role of ABA: does it mediate stress tolerance or plant growth retardation under long-term drought stress?

Recent developments in defining the functional basis of abscisic acid in regulating growth, development and stress response have provided essential components for its actions. We are yet to envision the impact of how differential levels of ABA influence plant growth across life cycle. Here we reviewed the information arising from the recent unprecedented advancement made in the field of ABA signaling operative under calcium-dependent and calcium-independent pathways mediating the transcriptional reprogramming under short-term stress response. Advancement made in the field of ABA receptors and transporters has started to fill major gaps in our understanding of the ABA action. However, ABA just not only regulates guard cell movement but impacts other reproductive tissue development through massive transcriptional reprogramming events affecting various stages of the plant life cycle. Therefore many questions still remain unanswered. One such intriguing question is the contradictory role of ABA known to mediate two opposite faces of the coin: regulating abiotic stress tolerance and imparting growth retardation. In this review, we critically assessed the impact of substantial elevated levels of ABA on impairment of photosynthesis and growth alteration and its subsequent influence on seed yield formation. Excess biosynthesis of ABA under stress may deprive the same precursor pool necessary for chlorophyll biosynthesis pathway, thereby triggering growth retardation. Further, we emphasized the importance of ABA homeostasis for integrating stress cues towards coordinating sustainable plant growth. Also we provided a pertinent background on ABA biosynthesis and degradation pathway manipulation to highlight the genes and processes used in genetic engineering of plants for changed ABA content.

Importance of ABA homeostasis under terminal drought stress in regulating grain filling events.

Recent studies suggest that abscisic acid (ABA) at its basal level plays an important role during seed set and grain filling events. Under drought stress ABA levels were found to be significantly enhanced in the developing seed. Until now we lack an understanding of (A) ABA homeostasis in developing seeds under terminal drought and (B) the interactive role of ABA in regulating the starch biosynthesis pathway in developing grains under terminal drought. We have recently reported the possible regulation of ABA homeostasis in source (flag leaf) and sink (developing grains) tissues under post-anthesis drought stress in barley and concluded that significantly enhanced ABA levels in developing grains are due to strong activation of the ABA deconjugation pathway and fine regulation of the ABA biosynthesis-degradation pathway.1 Additionally, we provided evidence for the role of ABA in differential regulation of starch biosynthesis genes and a significant upregulation of starch degradation beta amylase genes under drought, i.e. ABA not only influences the rate of starch accumulation but also starch quality.

Unbiased transcriptional comparisons of generalist and specialist herbivores feeding on progressively defenseless Nicotiana attenuata plants.

BACKGROUND: Herbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We created cDNA microarrays for Manduca sexta and Heliothis virescens separately, by spotting normalized midgut-specific cDNA libraries created from larvae that fed for 24 hours on MeJA-elicited wild-type (WT) Nicotiana attenuata plants. These microarrays were hybridized with labeled probes from neonates that fed for 24 hours on WT and isogenic plants progressively silenced in JA-mediated defenses (N: nicotine; N/PI: N and trypsin protease inhibitors; JA: all JA-mediated defenses). H. virescens neonates regulated 16 times more genes than did M. sexta neonates when they fed on plants silenced in JA-mediated defenses, and for both species, the greater the number of defenses silenced in the host plant (JA > N/PI > N), the greater were the number of transcripts regulated in the larvae. M. sexta larvae tended to down-regulate while H. virescens larvae up- and down-regulated transcripts from the same functional categories of genes. M. sexta larvae regulated transcripts in a diet-specific manner, while H. virescens larvae regulated a similar suite of transcripts across all diet types. CONCLUSIONS/SIGNIFICANCE: The observations are consistent with the expectation that specialists are better adapted than generalist herbivores to the defense responses elicited in their host plants by their feeding. While M. sexta larvae appear to be better adapted to N. attenuata's defenses, some of the elicited responses remain effective defenses against both herbivore species. The regulated genes provide novel insights into larval adaptations to N. attenuata's induced defenses, and represent potential targets for plant-mediated RNAi to falsify hypotheses about the process of adaptation.

Functional characterization of Nicotiana benthamiana homologs of peanut water deficit-induced genes by virus-induced gene silencing.

Determining the functional role of genes that are differentially regulated during a stress response is challenging. In this study, few water deficit-induced genes from peanut were characterized in Nicotiana benthamiana using virus-induced gene silencing (VIGS) and their relevance for stress adaptation was validated. Twenty-five cDNA clones from peanut water deficit stress-induced cDNA library that had more than 50% nucleotide similarity with N. benthamiana or tomato homologs were selected. VIGS in peanut is not yet feasible and therefore we characterized these 25 genes in N. benthamiana. Increased membrane damage was seen under water deficit stress in most of the silenced plants signifying that many of these stress-induced genes are important to confer drought tolerance. Among the genes tested, silencing by homolog of flavonol 3-O-glucosyltransferase (F3OGT), homolog of alcohol dehydrogenase, homologous to salt inducible protein, and homolog of heat shock protein 70 showed more visible wilting symptoms compared with the control plants during water deficit stress. Interestingly, down-regulation of two genes, homologous to aspartic proteinase 2, and homolog of Jumonji class of transcription factor showed relative drought tolerant phenotypes. F3OGT silenced plants showed more wilting symptoms, membrane damage and chlorophyll degradation than any other silenced plants during water deficit. Our results demonstrate that VIGS approach can be used to characterize and assess the functional relevance of water deficit stress-induced cDNAs in a heterologous species.