Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Transcriptome Analysis of Heat Shock Factor C2a Over-Expressing Wheat Roots Reveals Ferroptosis-like Cell Death in Heat Stress Recovery.

Wheat (Triticum aestivum L.) growing areas in many regions of the world are subject to heat waves which are predicted to increase in frequency because of climate change. The engineering of crop plants can be a useful strategy to mitigate heat stress-caused yield losses. Previously, we have shown that heat shock factor subclass C (TaHsfC2a-B)-overexpression significantly increased the survival of heat-stressed wheat seedlings. Although previous studies have shown that the overexpression of Hsf genes enhanced the survival of plants under heat stress, the molecular mechanisms are largely unknown. To understand the underlying molecular mechanisms involved in this response, a comparative analysis of the root transcriptomes of untransformed control and TaHsfC2a-overexpressing wheat lines by RNA-sequencing have been performed. The results of RNA-sequencing indicated that the roots of TaHsfC2a-overexpressing wheat seedlings showed lower transcripts of hydrogen peroxide-producing peroxidases, which corresponds to the reduced accumulation of hydrogen peroxide along the roots. In addition, suites of genes from iron transport and nicotianamine-related gene ontology categories showed lower transcript abundance in the roots of TaHsfC2a-overexpressing wheat roots than in the untransformed control line following heat stress, which are in accordance with the reduction in iron accumulation in the roots of transgenic plants under heat stress. Overall, these results suggested the existence of ferroptosis-like cell death under heat stress in wheat roots, and that TaHsfC2a is a key player in this mechanism. To date, this is the first evidence to show that a Hsf gene plays a key role in ferroptosis under heat stress in plants. In future, the role of Hsf genes could be further studied on ferroptosis in plants to identify root-based marker genes to screen for heat-tolerant genotypes.

Overexpression of TaCML20, a calmodulin-like gene, enhances water soluble carbohydrate accumulation and yield in wheat.

Calcium (Ca2+ ) is a universal messenger that mediates intracellular responses to extracellular stimuli in living organisms. Calmodulin (CaM) and calmodulin-like (CML) proteins are the important Ca2+ sensors in plants that decode Ca2+ -signatures to execute downstream intracellular level responses. Several studies indicate the interlinking of Ca2+ and sugar signaling in plants; however, no genes have been functionally characterized to provide molecular evidence. Our study found that expression of TaCML20 was significantly correlated with water soluble carbohydrate (WSC) concentrations in recombinant inbred lines in wheat. TaCML20 has four EF-hand motifs that may facilitate the binding of Ca2+ . To explore the role of CML20, we generated TaCML20 overexpressing transgenic lines in wheat. These lines accumulated higher WSC concentrations in the shoots, and we also found a significantly increased transcript level of sucrose:sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. In addition, TaCML20 overexpressing plants showed significantly increased tillers per plant and also increased about 19% of grain weight per plant compared with control plants. The results also suggested a role for TaCML20 in drought stress, as its transcripts significantly increased in the shoots of wild-type plants under water deficit. These results uncovered the role of CML20 in determining multiple traits in wheat.

Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway.

High temperature at grain filling can severely reduce wheat yield. Heat shock factors (Hsfs) are central regulators in heat acclimation. This study investigated the role of TaHsfC2a, a member of the monocot-specific HsfC2 subclass, in the regulation of heat protection genes in Triticum aestivum. Three TaHsfC2a homoeologous genes were highly expressed in wheat grains during grain filling and showed only transient up-regulation in the leaves by heat stress but were markedly up-regulated by drought and abscisic acid (ABA) treatment. Overexpression of TaHsfC2a-B in transgenic wheat resulted in up-regulation of a suite of heat protection genes (e.g. TaHSP70d and TaGalSyn). Most TaHsfC2a-B target genes were heat, drought and ABA inducible. Transactivation analysis of two representative targets (TaHSP70d and TaGalSyn) showed that TaHsfC2a-B activated expression of reporters driven by these target promoters. Promoter mutagenesis analyses revealed that heat shock element is responsible for transactivation by TaHsfC2a-B and heat/drought induction. TaHsfC2a-B-overexpressing wheat showed improved thermotolerance but not dehydration tolerance. Most TaHsfC2a-B target genes were co-up-regulated in developing grains with TaHsfC2a genes. These data suggest that TaHsfC2a-B is a transcriptional activator of heat protection genes and serves as a proactive mechanism for heat protection in developing wheat grains via the ABA-mediated regulatory pathway.

Chemical Management Strategies of Pimelea trichostachya Lindl. Using Pre- and Post-Emergence Herbicides.

Pimelea trichostachya Lindl. is a native Australian forb responsible for livestock poisoning and reducing the productivity and sustainability of grazing enterprises. This study was conducted as a pot trial under controlled conditions to investigate an effective chemical management strategy for P. trichostachya, a method that did not leave standing dead plant material, as such material can also be toxic to grazing cattle. Three herbicides, including one pre-emergence (tebuthiuron) and two post-emergence herbicides (2,4-D and metsulfuron-methyl), were tested in pot trials for their efficacy on P. trichostachya. Results showed that tebuthiuron applied as either a granular (10% active ingredient, a.i.) or pelleted (20% a.i.) form efficiently reduced the emergence of P. trichostachya seedlings. Although some seedlings emerged, they perished within 7 days post treatment, leaving no residual plant matter. Testing now needs to be undertaken under field conditions to validate the findings within vegetation communities where potential non-target impacts need to be accounted for as well. The post-emergence application of 2,4-D and metsulfuron-methyl demonstrated that the highest efficacy and reduced application rates were achieved by treating earlier growth stages (i.e., seedlings) of P. trichostachya plants. In addition, the amount of toxic dead plant material was minimized due to the faster degradation of these small plants. These findings offer practical, cost-effective solutions for sustaining grazing lands from P. trichostachya challenges.

Environmental Effects during Early Life-History Stages and Seed Development on Seed Functional Traits of an Australian Native Legume Species.

Understanding how seed functional traits interact with environmental factors to determine seedling recruitment is critical to assess the impact of climate change on ecosystem restoration. This study focused on the effects of environmental factors on the mother plant during early plant life history stages and during seed development. Desmodium brachypodum A. Gray (large tick trefoil, Fabaceae) was used as a model species. Firstly, this study analyzed seed germination traits in response to temperature and moisture stress. Secondly, it investigated how seed burial depth interacts with temperature and soil moisture to influence seedling emergence traits. Finally, it determined if contrasting levels of post-anthesis soil moisture could result in changes in D. brachypodum reproductive biology and seed and seedling functional traits. The results showed that elevated temperature and moisture stress interacted to significantly reduce the seed germination and seedling emergence (each by >50%), while the seed burial improved the seedling emergence. Post-anthesis soil moisture stress negatively impacted the plant traits, reducing the duration of the reproductive phenology stage (by 9 days) and seed production (by almost 50%). Unexpectedly, soil moisture stress did not affect most seed or seedling traits. In conclusion, elevated temperatures combined with low soil moisture caused significant declines in seed germination and seedling emergence. On the other hand, the reproductive output of D. brachypodum had low seed variability under soil moisture stress, which might be useful when sourcing seeds from climates with high variability. Even so, a reduction in seed quantity under maternal moisture stress can impact the long-term survival of restored plant populations.

Towards Pathogen-Free Coconut Germplasm Exchange.

Coconut (Cocos nucifera L.) is an important palm species that serves as the mainstay of several industries and contributes to the livelihoods of millions of smallholder farmers. International exchange of coconut germplasm has been undertaken for several decades to facilitate the conservation of selected varieties within global genebanks and for the distribution to farmers and scientists. In vitro systems are a convenient and an efficient method for the exchange of coconut germplasm. However, it is possible that these tissue culture systems can transfer lethal pathogens causing a threat to the importing countries. In this review, the following topics are discussed: the major disease-causing agents of concern, the various tissues that could be used for coconut germplasm exchange, and the techniques available for the detection and elimination of disease-causing agents from various transmission systems. Additionally, the lack of clear, science-backed guidelines to facilitate the exchange of in vitro coconut materials is raised, along with recommendations for future studies to ensure the safe movement of coconut germplasm without biosecurity risks.

Coconut Callus Initiation for Cell Suspension Culture.

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species.

Chemical Management of Senecio madagascariensis (Fireweed).

Fireweed (Senecio madagascariensis Poir.) is a herbaceous weed-producing pyrrolizidine alkaloid that is poisonous to livestock. To investigate the efficacy of chemical management on fireweed and its soil seed bank density, a field experiment was conducted in Beechmont, Queensland, in 2018 within a pasture community. A total of four herbicides (bromoxynil, fluroxypyr/aminopyralid, metsulfuron-methyl and triclopyr/picloram/aminopyralid) were applied either singularly or repeated after 3 months to a mix-aged population of fireweed. The initial fireweed plant density at the field site was high (10 to 18 plants m-2). However, after the first herbicide application, the fireweed plant density declined significantly (to ca. 0 to 4 plants m-2), with further reductions following the second treatment. Prior to herbicide application, fireweed seeds in both the upper (0 to 2 cm) and lower (2 to 10 cm) soil seed bank layers averaged 8804 and 3593 seeds m-2, respectively. Post-herbicide application, the seed density was significantly reduced in both the upper (970 seeds m-2) and lower (689 seeds m-2) seed bank layers. Based on the prevailing environmental conditions and nil grazing strategy of the current study, a single application of either fluroxypyr/aminopyralid, metsulfuron-methyl or triclopyr/picloram/aminopyralid would be sufficient to achieve effective control, whilst a second follow-up application is required with bromoxynil.

Factors Affecting the Germination Ecology of Pimelea trichostachya and Its Relationship to Field Emergence.

Pimelea trichostachya Lindl is a little-understood Australian native plant, with irregular field emergence, causing significant poisoning to grazing livestock. The study aims to examine the form of dormancy exhibited by P. trichostachya and determine how key environmental conditions, such as alternating temperature and light conditions, moisture availability, substrate pH and burial depth, affect its germination and emergence. The study concludes that P. trichostachya has a complex dormancy mechanism. This comprises a physical component that can be partly removed by fruit scarification, a metabolic dormancy that can be overcome by gibberellic acid (GA3), and a suspected third mechanism based on a water-soluble germination inhibitor. The results showed that scarified single seeded fruit (hereafter seed) with GA3 treatment gave the highest germination percentage (86 ± 3%) at 25/15 °C, with good germination rates at other temperature regimes. Light exposure stimulated germination, but a significant proportion of seeds still germinated in the dark. The study also found that seeds could germinate under water-limited conditions and a wide range of pH levels (4 to 8). Seedling emergence was inhibited when seeds were buried below 3 cm in soil. Pimelea trichostachya emergence in the field commonly occurs from Autumn to Spring. Understanding its dormancy mechanism and recognizing its triggers for germination will enable better prediction of outbreaks. This can help landholders prepare for emergence and help manage seedbank build-up in pastures and crops.

Poisonous Plants of the Genus Pimelea: A Menace for the Australian Livestock Industry.

Pimelea is a genus of about 140 plant species, some of which are well-known for causing animal poisoning resulting in significant economic losses to the Australian livestock industry. The main poisonous species/subspecies include Pimelea simplex (subsp. simplex and subsp. continua), P. trichostachya and P. elongata (generally referred to as Pimelea). These plants contain a diterpenoid orthoester toxin, called simplexin. Pimelea poisoning is known to cause the death of cattle (Bos taurus and B. indicus) or weaken surviving animals. Pimelea species are well-adapted native plants, and their diaspores (single seeded fruits) possess variable degrees of dormancy. Hence, the diaspores do not generally germinate in the same recruitment event, which makes management difficult, necessitating the development of integrated management strategies based on infestation circumstances (e.g., size and density). For example, the integration of herbicides with physical control techniques, competitive pasture establishment and tactical grazing could be effective in some situations. However, such options have not been widely adopted at the field level to mitigate ongoing management challenges. This systematic review provides a valuable synthesis of the current knowledge on the biology, ecology, and management of poisonous Pimelea species with a focus on the Australian livestock industry while identifying potential avenues for future research.

Fruit Biology of Coconut (Cocos nucifera L.).

Coconut (Cocos nucifera L.) is an important perennial crop adapted to a wide range of habitats. Although global coconut demand has increased sharply over the past few years, its production has been decreasing due to palm senility, as well as abiotic and biotic stresses. In fact, replanting efforts are impeded due to the lack of good quality seedlings. In vitro technologies have a great potential; however, their applications may take time to reach a commercial level. Therefore, traditional seed propagation is still critical to help meet the rising demand and its practice needs to be improved. To achieve an improved propagation via seeds, it is important to understand coconut fruit biology and its related issues. This review aims to provide a comprehensive summary of the existing knowledge on coconut fruit morpho-anatomy, germination biology, seed dispersal, distribution, fruit longevity and storage. This will help to identify gaps where future research efforts should be directed to improve traditional seed propagation.

Cloning Coconut via Somatic Embryogenesis: A Review of the Current Status and Future Prospects.

Coconut [Cocos nucifera L.] is often called "the tree of life" because of its many uses in the food, beverage, medicinal, and cosmetic industries. Currently, more than 50% of the palms grown throughout the world are senile and need to be replanted immediately to ensure production levels meet the present and increasing demand for coconut products. Mass replanting will not be possible using traditional propagation methods from seed. Recent studies have indicated that in vitro cloning via somatic embryogenesis is the most promising alternative for the large-scale production of new coconut palms. This paper provides a review on the status and prospects for the application of somatic embryogenesis to mass clonal propagation of coconut.

Ferroptosis: Yet Another Way to Die.

Cell death is one of the most fundamental biological processes operating in multicellular organisms. Recent research highlighted here [Distéfano et al. (J. Cell Biol. 2017:216;463-476) and Dangol et al. (Plant Cell 2019:31;189-209)] revealed an iron- and ROS-dependent cell death phenomenon called ferroptosis in plants. Features distinguishing ferroptosis from other cell death events and how ferroptosis can be exploited to improve plant performance are discussed.

Rapid and Quantitative CELD Assay to Measure the Specificity of Transcription Factor-DNA-Binding Interactions and Identify cis-Elements.

Specific interaction between transcription factor (TF) and cis-regulatory elements in the context of chromatin is the key to determining gene expression. Thus, it is important to measure DNA-binding specificity and identify cis-elements of TFs of your interest. In this chapter, we described a microwell-based assay to determine DNA-binding specificity by using translational fusion of a TF with a highly active cellulase (CELD), which hydrolyzes 4-methylumbelliferyl ß-D-cellobioside to a fluorescent 4-methylumbelliferone product. The hydrolysis activity arrows us to quantify the binding strength between TF-CELD fusion protein and biotinylated DNA sequences in a 96-well microplate. The high-throughput and quantitative nature of CELD assay enable researchers to test a large number of putative DNA-binding sites of TFs, which subsequently leads to the identification of its direct target genes.

Identification and Functional Characterization of Sugarcane Invertase Inhibitor (ShINH1): A Potential Candidate for Reducing Pre- and Post-harvest Loss of Sucrose in Sugarcane.

In sugarcane, invertase enzymes play a key role in sucrose accumulation and are also involved in futile reactions where sucrose is continuously degraded during the pre- and post-harvest period, thereby reducing sugar yield and recovery. Invertase inhibitor (INVINH) proteins play a key role in post-translation regulation of plant invertases through which sucrose hydrolysis is controlled. INVINH proteins are small (18 kDa) members of the pectin methylesterase inhibitor superfamily and they are moderately conserved across plants. In the present study, we identified two INVINH genes from sugarcane, ShINH1 and ShINH2. In silico characterization of the encoded proteins revealed 43% sequence identity at the amino acid level, confirming the non-allelic nature of the proteins. The presence of putative signal peptide and subcellular targeting sequences revealed that ShINH1 and ShINH2 likely have apoplasmic and vacuolar localization, respectively. Experimental visualization of ShINH1-GFP revealed that ShINHI is indeed exported to the apoplast. Differential tissue-specific and developmental expression of ShINH1 between leaf, stalk, flower and root suggest that it plays a role in controlling source-sink metabolic regulation during sucrose accumulation in sugarcane. ShINH1 is expressed at relatively high levels in leaves and stalk compared to flowers and roots, and expression decreases significantly toward internodal maturity during stalk development. ShINH1 is expressed at variable levels in flowers with no specific association to floral maturity. Production of recombinant ShINH1 enabled experimental validation of protein function under in vitro conditions. Recombinant ShINH1 potently inhibited acid invertase (IC50 22.5 nM), making it a candidate for controlling pre- and post-harvest deterioration of sucrose in sugarcane. Our results indicate that ShINH1 and ShINH2 are likely to play a regulatory role in sucrose accumulation and contribute to the improvement of sugar yield and recovery in sugarcane.