Genomic analysis reveals phylogeny of Zygophyllales and mechanism for water retention of a succulent xerophyte.

Revealing the genetic basis for stress-resistant traits in extremophile plants will yield important information for crop improvement. Zygophyllum xanthoxylum, an extant species of the ancient Mediterranean, is a succulent xerophyte that can maintain a favorable water status under desert habitats; however, the genetic basis of this adaptive trait is poorly understood. Furthermore, the phylogenetic position of Zygophyllales, to which Z. xanthoxylum belongs, remains controversial. In this study, we sequenced and assembled the chromosome-level genome of Z. xanthoxylum. Phylogenetic analysis showed that Zygophyllales and Myrtales form a separated taxon as a sister to the clade comprising fabids and malvids, clarifying the phylogenetic position of Zygophyllales at whole-genome scale. Analysis of genomic and transcriptomic data revealed multiple critical mechanisms underlying the efficient osmotic adjustment using Na+ and K+ as "cheap" osmolytes that Z. xanthoxylum has evolved through the expansion and synchronized expression of genes encoding key transporters/channels and their regulators involved in Na+/K+ uptake, transport, and compartmentation. It is worth noting that ZxCNGC1;1 (cyclic nucleotide-gated channels) and ZxCNGC1;2 constituted a previously undiscovered energy-saving pathway for Na+ uptake. Meanwhile, the core genes involved in biosynthesis of cuticular wax also featured an expansion and upregulated expression, contributing to the water retention capacity of Z. xanthoxylum under desert environments. Overall, these findings boost the understanding of evolutionary relationships of eudicots, illustrate the unique water retention mechanism in the succulent xerophyte that is distinct from glycophyte, and thus provide valuable genetic resources for the improvement of stress tolerance in crops and insights into the remediation of sodic lands.

Ca2+/calmodulin-mediated desensitization of glutamate receptors shapes plant systemic wound signalling and anti-herbivore defence.

Plants rely on systemic signalling mechanisms to establish whole-body defence in response to insect and nematode attacks. GLUTAMATE RECEPTOR-LIKE (GLR) genes have been implicated in long-distance transmission of wound signals to initiate the accumulation of the defence hormone jasmonate (JA) at undamaged distal sites. The systemic signalling entails the activation of Ca2+-permeable GLR channels by wound-released glutamate, triggering membrane depolarization and cytosolic Ca2+ influx throughout the whole plant. The systemic electrical and calcium signals rapidly dissipate to restore the resting state, partially due to desensitization of the GLR channels. Here we report the discovery of calmodulin-mediated, Ca2+-dependent desensitization of GLR channels, revealing a negative feedback loop in the orchestration of plant systemic wound responses. A CRISPR-engineered GLR3.3 allele with impaired desensitization showed prolonged systemic electrical signalling and Ca2+ waves, leading to enhanced plant defence against herbivores. Moreover, this Ca2+/calmodulin-mediated desensitization of GLR channels is a highly conserved mechanism in plants, providing a potential target for engineering anti-herbivore defence in crops.

RALF signaling pathway activates MLO calcium channels to maintain pollen tube integrity.

Pollen tube tip growth requires intricate Ca2+ signaling. Recent studies have also identified rapid alkalization factor (RALF)-family peptides and their receptors as critical components for pollen tube tip growth and integrity. The functional relationship of RALF and calcium signaling modules remains largely unclear. Here we report that disruption of RALF signaling pathway abolished the cytosolic Ca2+ gradient in the pollen tube, indicating that Ca2+ signaling is downstream of the RALF signaling pathway. We identified MILDEW RESISTANCE LOCUS O (MLO) family proteins MLO1, 5, 9, 15, as Ca2+ channels required for Ca2+ influx and pollen tube integrity. We further reconstituted the biochemical pathway in which signaling via RALF and RALF receptors activated MLO1/5/9/15 calcium channels. Together, we conclude that RALF peptides derived from pollen tube bind to their receptors to establish pollen tube Ca2+ gradient through activation of the MLO channels. Our finding has thus provided a mechanistic link between the RALF signaling pathway and Ca2+ signaling in controlling pollen tube integrity and growth.

A receptor-channel trio conducts Ca2+ signalling for pollen tube reception.

Precise signalling between pollen tubes and synergid cells in the ovule initiates fertilization in flowering plants1. Contact of the pollen tube with the ovule triggers calcium spiking in the synergids2,3 that induces pollen tube rupture and sperm release. This process, termed pollen tube reception, entails the action of three synergid-expressed proteins in Arabidopsis: FERONIA (FER), a receptor-like kinase; LORELEI (LRE), a glycosylphosphatidylinositol-anchored protein; and NORTIA (NTA), a transmembrane protein of unknown function4-6. Genetic analyses have placed these three proteins in the same pathway; however, it remains unknown how they work together to enable synergid-pollen tube communication. Here we identify two pollen-tube-derived small peptides7 that belong to the rapid alkalinization factor (RALF) family8 as ligands for the FER-LRE co-receptor, which in turn recruits NTA to the plasma membrane. NTA functions as a calmodulin-gated calcium channel required for calcium spiking in the synergid. We also reconstitute the biochemical pathway in which FER-LRE perceives pollen-tube-derived peptides to activate the NTA calcium channel and initiate calcium spiking, a second messenger for pollen tube reception. The FER-LRE-NTA trio therefore forms a previously unanticipated receptor-channel complex in the female cell to recognize male signals and trigger the fertilization process.

Peroxymonosulfate activation by Co-doped magnetic Mn3O4 for degradation of oxytetracycline in water.

Co-doped magnetic Mn3O4 was synthesized by the solvothermal method and adopted as an effective catalyst for the degradation of oxytetracycline (OTC) in water. Synergistic interactions between Co-Mn3O4 and Fe3O4 not only resulted in the enhanced catalytic activity through the activation of peroxymonosulfate (PMS) to degrade OTC but also made Fe3O4/Co-Mn3O4 easy to be separated and recovered from aqueous solution. 94.2% of OTC could be degraded within 60 min at an initial OTC concentration of 10 mg L-1, catalyst dosage of 0.2 g L-1, and PMS concentration of 10 mM. The high efficiency of OTC removal was achieved in a wider pH range of 3.0-10.0. Co (II), Co (III), Fe (II), Fe (III), Mn (II), Mn (III), and Mn (IV) on Fe3O4/Co-Mn3O4 were identified as catalytic sites based on XPS analysis. The free radical quenching experiments showed that O2•- radicals and 1O2 played the main role in the degradation process and the catalytic degradation of OTC involved both free radical and non-free radical reactions. Eventually, the intermediates of OTC degradation were examined, and the possible decomposition pathways were proposed. The excellent catalytic performances of Fe3O4/Co-Mn3O4 came from the fact that the large specific surface area could provide abundant active sites for the activation of PMS and the redistribution of inter-atomic charges accelerated the redox reactions of metal ions. The high degradation efficiency and rate constant of OTC in actual water samples indicated that Fe3O4/Co-Mn3O4 had a good practical application potential.

A plasma membrane transporter coordinates phosphate reallocation and grain filling in cereals.

Phosphate (Pi) is essential to plant growth and crop yield. However, it remains unknown how Pi homeostasis is maintained during cereal grain filling. Here, we identified a rice grain-filling-controlling PHO1-type Pi transporter, OsPHO1;2, through map-based cloning. Pi efflux activity and its localization to the plasma membrane of seed tissues implicated a specific role for OsPHO1;2 in Pi reallocation during grain filling. Indeed, Pi over-accumulated in developing seeds of the Ospho1;2 mutant, which inhibited the activity of ADP-glucose pyrophosphorylase (AGPase), important for starch synthesis, and the grain-filling defect was alleviated by overexpression of AGPase in Ospho1;2-mutant plants. A conserved function was recognized for the maize transporter ZmPHO1;2. Importantly, ectopic overexpression of OsPHO1;2 enhanced grain yield, especially under low-Pi conditions. Collectively, we discovered a mechanism underlying Pi transport, grain filling and P-use efficiency, providing an efficient strategy for improving grain yield with minimal P-fertilizer input in cereals.

Recent Advances in Genome-wide Analyses of Plant Potassium Transporter Families.

Plants require potassium (K+) as a macronutrient to support numerous physiological processes. Understanding how this nutrient is transported, stored, and utilized within plants is crucial for breeding crops with high K+ use efficiency. As K+ is not metabolized, cross-membrane transport becomes a rate-limiting step for efficient distribution and utilization in plants. Several K+ transporter families, such as KUP/HAK/KT and KEA transporters and Shaker-like and TPK channels, play dominant roles in plant K+ transport processes. In this review, we provide a comprehensive contemporary overview of our knowledge about these K+ transporter families in angiosperms, with a major focus on the genome-wide identification of K+ transporter families, subcellular localization, spatial expression, function and regulation. We also expanded the genome-wide search for the K+ transporter genes and examined their tissue-specific expression in Camelina sativa, a polyploid oil-seed crop with a potential to adapt to marginal lands for biofuel purposes and contribution to sustainable agriculture. In addition, we present new insights and emphasis on the study of K+ transporters in polyploids in an effort to generate crops with high K+ Utilization Efficiency (KUE).

Two glutamate- and pH-regulated Ca2+ channels are required for systemic wound signaling in Arabidopsis.

Plants defend against herbivores and nematodes by rapidly sending signals from the wounded sites to the whole plant. We investigated how plants generate and transduce these rapidly moving, long-distance signals referred to as systemic wound signals. We developed a system for measuring systemic responses to root wounding in Arabidopsis thaliana We found that root wounding or the application of glutamate to wounded roots was sufficient to trigger root-to-shoot Ca2+ waves and slow wave potentials (SWPs). Both of these systemic signals were inhibited by either disruption of both GLR3.3 and GLR3.6, which encode glutamate receptor-like proteins (GLRs), or constitutive activation of the P-type H+-ATPase AHA1. We further showed that GLR3.3 and GLR3.6 displayed Ca2+-permeable channel activities gated by both glutamate and extracellular pH. Together, these results support the hypothesis that wounding inhibits P-type H+-ATPase activity, leading to apoplastic alkalization. This, together with glutamate released from damaged phloem, activates GLRs, resulting in depolarization of membranes in the form of SWPs and the generation of cytosolic Ca2+ increases to propagate systemic wound signaling.

Calcium spikes, waves and oscillations in plant development and biotic interactions.

The calcium ion (Ca2+) is a universal signal in all eukaryotic cells. A fundamental question is how Ca2+, a simple cation, encodes complex information with high specificity. Extensive research has established a two-step process (encoding and decoding) that governs the specificity of Ca2+ signals. While the encoding mechanism entails a complex array of channels and transporters, the decoding process features a number of Ca2+ sensors and effectors that convert Ca2+ signals into cellular effects. Along this general paradigm, some signalling components may be highly conserved, but others are divergent among different organisms. In plant cells, Ca2+ participates in numerous signalling processes, and here we focus on the latest discoveries on Ca2+-encoding mechanisms in development and biotic interactions. In particular, we use examples such as polarized cell growth of pollen tube and root hair in which tip-focused Ca2+ oscillations specify the signalling events for rapid cell elongation. In plant-microbe interactions, Ca2+ spiking and oscillations hold the key to signalling specificity: while pathogens elicit cytoplasmic spiking, symbiotic microorganisms trigger nuclear Ca2+ oscillations. Herbivore attacks or mechanical wounding can trigger Ca2+ waves traveling a long distance to transmit and convert the local signal to a systemic defence program in the whole plant. What channels and transporters work together to carve out the spatial and temporal patterns of the Ca2+ fluctuations? This question has remained enigmatic for decades until recent studies uncovered Ca2+ channels that orchestrate specific Ca2+ signatures in each of these processes. Future work will further expand the toolkit for Ca2+-encoding mechanisms and place Ca2+ signalling steps into larger signalling networks.

Dynamic Interactions of Plant CNGC Subunits and Calmodulins Drive Oscillatory Ca2+ Channel Activities.

Calcium is a universal signal in all eukaryotes, but the mechanism for encoding calcium signatures remains largely unknown. Calcium oscillations control pollen tube growth and fertilization in flowering plants, serving as a model for dissecting the molecular machines that mediate calcium fluctuations. We report that pollen-tube-specific cyclic nucleotide-gated channels (CNGC18, CNGC8, and CNGC7) together with calmodulin 2 (CaM2) constitute a molecular switch that either opens or closes the calcium channel depending on cellular calcium levels. Under low calcium, calcium-free calmodulin 2 (Apo-CaM2) interacts with CNGC18-CNGC8 complex, leading to activation of the influx channel and consequently increasing cytosolic calcium levels. Calcium-bound CaM2 dissociates from CNGC18/8 heterotetramer, closing the channel and initiating a downturn of cellular calcium levels. We further reconstituted the calcium oscillator in HEK293 cells, supporting the model that Ca2+-CaM-dependent regulation of CNGC channel activity provides an auto-regulatory feedback mechanism for calcium oscillations during pollen tube growth.

Cyclic nucleotide-gated channel 18 functions as an essential Ca2+ channel for pollen germination and pollen tube growth in Arabidopsis.

We recently revealed that cyclic nucleotide-gated channel 18 (CNGC18) functioned as the main Ca2+ channel in pollen tube tips for pollen tube guidance to ovules by regulating external Ca2+ influx in Arabidopsis. In this study, we found that the reduction of external Ca2+ concentration ([Ca2+]ext) from 10 mM to 5 mM, and further to 2 mM, led to the decreases of pollen germination percentages, but led to the increases of the percentages of ruptured pollen grains and tubes, and branched pollen tubes in vitro in cngc18-17 compared with wild type. The second point mutant allele cngc18-22 showed similar phenotypes, including reduced pollen germination percentages, increased percentages of ruptured pollen tubes, but did not show obvious different percentages of ruptured pollen grains and branched pollen tubes compared with wild type. These data demonstrate that CNGC18 plays essential roles in pollen germination and tube growth as a Ca2+ channel in Arabidopsis.

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance in Arabidopsis.

Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrate-selective in rice.

MAIN CONCLUSION: OsSAPK8 is an essential activator of OsSLAC1 by phosphorylation, and OsSLAC1 is a nitrate-selective anion channel. S-type anion channel AtSLAC1 and protein kinase AtOST1 have been well-characterized as two core components of ABA signaling cascade in Arabidopsis guard cells, and AtOST1 functions as a main upstream activator of AtSLAC1 for drought stress- and ABA-induced stomata closure. However, the identity of the ortholog of AtOST1 in rice, the main activator of OsSLAC1, is still unknown. Here, we report that protein kinase OsSAPK8 interacts with and activates OsSLAC1 mainly by phosphorylating serine 129 (S129) of OsSLAC1, and this phosphorylating site corresponds to the specific phosphorylating site serine 120 (S120) of AtSLAC1 for AtOST1. Additionally, we found that OsSLAC1 is a nitrate-selective anion channel without obvious permeability to chloride, malate, and sulfate, and the expression of OsSLAC1 in Arabidopsis slac1-3 (atslac1-3) mutant successfully rescued the hypersensitive phenotype of this mutant to drought stress. Together, this research suggests that OsSAPK8 is a counterpart of AtOST1 for the activation of OsSLAC1, which is a nitrate-selective anion channel.