The plant immune system is well equipped to ward off the attacks of different types of phytopathogens. It primarily relies on two types of immune sensors-plasma membrane-resident receptor-like kinases and intracellular nucleotide-binding domain leucine-rich repeat (NLRs) receptors that engage preferentially in pattern- and effector-triggered immunity, respectively. Delicate fine-tuning, in particular of the NLR-governed branch of immunity, is key to prevent inappropriate and deleterious activation of plant immune responses. Inadequate NLR allele constellations, such as in the case of hybrid incompatibility, and the mis-activation of NLRs or the absence or modification of proteins guarded by these NLRs can result in the spontaneous initiation of plant defense responses and cell death-a phenomenon referred to as plant autoimmunity. Here, we review recent insights augmenting our mechanistic comprehension of plant autoimmunity. The recent findings broaden our understanding regarding hybrid incompatibility, unravel candidates for proteins likely guarded by NLRs and underline the necessity for the fine-tuning of NLR expression at various levels to avoid autoimmunity. We further present recently emerged tools to study plant autoimmunity and draw a cross-kingdom comparison to the role of NLRs in animal autoimmune conditions.

Erratum in Plant Physiol. 2022 Jun 1;189(2):1169.

The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%-86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin-Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].

AGAMOUS-Like 18 (AGL18) is a MADS domain transcription factor (TF) that is structurally related to AGL15. Here we show that, like AGL15, AGL18 can promote somatic embryogenesis (SE) when ectopically expressed in Arabidopsis (Arabidopsis thaliana). Based on loss-of-function mutants, AGL15 and AGL18 have redundant functions in developmental processes such as SE. To understand the nature of this redundancy, we undertook a number of studies to look at the interaction between these factors. We studied the genome-wide direct targets of AGL18 to characterize its roles at the molecular level using chromatin immunoprecipitation (ChIP)-SEQ combined with RNA-SEQ. The results demonstrated that AGL18 binds to thousands of sites in the genome. Comparison of ChIP-SEQ data for AGL15 and AGL18 revealed substantial numbers of genes bound by both AGL15 and AGL18, but there were also differences. Gene ontology analysis revealed that target genes were enriched for seed, embryo, and reproductive development as well as hormone and stress responses. The results also demonstrated that AGL15 and AGL18 interact in a complex regulatory loop, where AGL15 inhibited transcript accumulation of AGL18, while AGL18 increased AGL15 transcript accumulation. Co-immunoprecipitation revealed an interaction between AGL18 and AGL15 in somatic embryo tissue. The binding and expression analyses revealed a complex crosstalk and interactions among embryo TFs and their target genes. In addition, our study also revealed that phosphorylation of AGL18 and AGL15 was crucial for the promotion of SE.

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

Predicting and understanding plant responses to perturbations require integrating the interactions between nutritional sources, genes, cell metabolism, and physiology in the same model. This can be achieved using metabolic modeling calibrated by experimental data. In this study, we developed a multi-organ metabolic model of a tomato (Solanum lycopersicum) plant during vegetative growth, named Virtual Young TOmato Plant (VYTOP) that combines genome-scale metabolic models of leaf, stem and root and integrates experimental data acquired from metabolomics and high-throughput phenotyping of tomato plants. It is composed of 6,689 reactions and 6,326 metabolites. We validated VYTOP predictions on five independent use cases. The model correctly predicted that glutamine is the main organic nutrient of xylem sap. The model estimated quantitatively how stem photosynthetic contribution impacts exchanges between the different organs. The model was also able to predict how nitrogen limitation affects vegetative growth and the metabolic behavior of transgenic tomato lines with altered expression of core metabolic enzymes. The integration of different components, such as a metabolic model, physiological constraints, and experimental data, generates a powerful predictive tool to study plant behavior, which will be useful for several other applications, such as plant metabolic engineering or plant nutrition.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

Uptake of boron (B) in rice (Oryza sativa) is mediated by the Low silicon rice 1 (OsLsi1) channel, belonging to the NOD26-like intrinsic protein III subgroup, and the efflux transporter B transporter 1 (OsBOR1). However, it is unknown how these transporters cooperate for B uptake and how they are regulated in response to B fluctuations. Here, we examined the response of these two transporters to environmental B changes at the transcriptional and posttranslational level. OsBOR1 showed polar localization at the proximal side of both the exodermis and endodermis of mature root region, forming an efficient uptake system with OsLsi1 polarly localized at the distal side of the same cell layers. Expression of OsBOR1 and OsLsi1 was unaffected by B deficiency and excess. However, although OsLsi1 protein did not respond to high B at the protein level, OsBOR1 was degraded in response to high B within hours, which was accompanied with a significant decrease of total B uptake. The high B-induced degradation of OsBOR1 was inhibited in the presence of MG-132, a proteasome inhibitor, without disturbance of the polar localization. In contrast, neither the high B-induced degradation of OsBOR1 nor its polarity was affected by induced expression of dominant-negative mutated dynamin-related protein 1A (OsDRP1AK47A) or knockout of the mu subunit (AP2M) of adaptor protein-2 complex, suggesting that clathrin-mediated endocytosis is not involved in OsBOR1 degradation and polar localization. These results indicate that, in contrast to Arabidopsis thaliana, rice has a distinct regulatory mechanism for B uptake through clathrin-independent degradation of OsBOR1 in response to high B.

In the wild cruciferous wintercress (Barbarea vulgaris), β-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce β-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of β-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Drought stress tolerance is a complex trait regulated by multiple factors. Here, we demonstrate that the miRNA160-Auxin Response Factor 17 (ARF17)-HYPONASTIC LEAVES 1 module is crucial for apple (Malus domestica) drought tolerance. Using stable transgenic plants, we found that drought tolerance was improved by higher levels of Mdm-miR160 or MdHYL1 and by decreased levels of MdARF17, whereas reductions in MdHYL1 or increases in MdARF17 led to greater drought sensitivity. Further study revealed that modulation of drought tolerance was achieved through regulation of drought-responsive miRNA levels by MdARF17 and MdHYL1; MdARF17 interacted with MdHYL1 and bound to the promoter of MdHYL1. Genetic analysis further suggested that MdHYL1 is a direct downstream target of MdARF17. Importantly, MdARF17 and MdHYL1 regulated the abundance of Mdm-miR160. In addition, the Mdm-miR160-MdARF17-MdHYL1 module regulated adventitious root development. We also found that Mdm-miR160 can move from the scion to the rootstock in apple and tomato (Solanum lycopersicum), thereby improving root development and drought tolerance of the rootstock. Our study revealed the mechanisms by which the positive feedback loop of Mdm-miR160-MdARF17-MdHYL1 influences apple drought tolerance.

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis (Arabidopsis thaliana) has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem elongates with the top leaf primordia developing into cauline leaves. Meristems in Arabidopsis initiate in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three Arabidopsis ecotypes and several Arabidopsis mutants with varied shoot architectures. Our results showed no negative correlation between cauline and rosette branch numbers in Arabidopsis, demonstrating that there is no tradeoff between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and Arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number of rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity, and temperature. Our data reveal different levels of plasticity in the regulation of branching at rosette and cauline nodes, and promote a framework for future branching analyses.

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, β-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues.

Proline (Pro) catabolism and reactive oxygen species production have been linked in mammals and Caenorhabditis elegans, while increases in leaf respiration rate follow Pro exposure in plants. Here, we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis (Arabidopsis thaliana) leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves displayed symptoms of elevated oxidative stress and suffered increased oxidative damage during Pro metabolism compared to the wild-type (WT) or the single mutants. During recovery from salt stress, when relatively high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than in the WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.

Mechanical properties, size and geometry of cells, and internal turgor pressure greatly influence cell morphogenesis. Computational models of cell growth require values for wall elastic modulus and turgor pressure, but very few experiments have been designed to validate the results using measurements that deform the entire thickness of the cell wall. New wall material is synthesized at the inner surface of the cell such that full-thickness deformations are needed to quantify relevant changes associated with cell development. Here, we present an integrated, experimental-computational approach to analyze quantitatively the variation of elastic bending behavior in the primary cell wall of living Arabidopsis (Arabidopsis thaliana) pavement cells and to measure turgor pressure within cells under different osmotic conditions. This approach used laser scanning confocal microscopy to measure the 3D geometry of single pavement cells and indentation experiments to probe the local mechanical responses across the periclinal wall. The experimental results were matched iteratively using a finite element model of the experiment to determine the local mechanical properties and turgor pressure. The resulting modulus distribution along the periclinal wall was nonuniform across the leaf cells studied. These results were consistent with the characteristics of plant cell walls which have a heterogeneous organization. The results and model allowed the magnitude and orientation of cell wall stress to be predicted quantitatively. The methods also serve as a reference for future work to analyze the morphogenetic behaviors of plant cells in terms of the heterogeneity and anisotropy of cell walls.

Efficient foraging by plant roots relies on the ability to sense multiple physical and chemical cues in soil and to reorient growth accordingly (tropism). Root tropisms range from sensing gravity (gravitropism), light (phototropism), water (hydrotropism), touch (thigmotropism), and more. Electrotropism, also known as galvanotropism, is the phenomenon of aligning growth with external electric fields and currents. Although root electrotropism has been observed in a few species since the end of the 19th century, its molecular and physical mechanisms remain elusive, limiting its comparison with the more well-defined sensing pathways in plants. Here, we provide a quantitative and molecular characterization of root electrotropism in the model system Arabidopsis (Arabidopsis thaliana), showing that it does not depend on an asymmetric distribution of the plant hormone auxin, but instead requires the biosynthesis of a second hormone, cytokinin. We also show that the dose-response kinetics of the early steps of root electrotropism follows a power law analogous to the one observed in some physiological reactions in animals. Future studies involving more extensive molecular and quantitative characterization of root electrotropism would represent a step toward a better understanding of signal integration in plants and would also serve as an independent outgroup for comparative analysis of electroreception in animals and fungi.

Tanshinone ⅡA (TⅡA), a diterpene quinone with a furan ring, is a bioactive compound found in the medicinal herb redroot sage (Salvia miltiorrhiza Bunge), in which both furan and dihydrofuran analogs are present in abundance. Progress has been made recently in elucidating the tanshinone biosynthetic pathway, including heterocyclization of the dihydrofuran D-ring by cytochrome P450s; however, dehydrogenation of dihydrofuran to furan, a key step of furan ring formation, remains uncharacterized. Here, by differential transcriptome mining, we identified six 2-oxoglutarate-dependent dioxygenase (2-ODD) genes whose expressions corresponded to tanshinone biosynthesis. We showed that Sm2-ODD14 acts as a dehydrogenase catalyzing the furan ring aromatization. In vitro Sm2-ODD14 converted cryptotanshinone to TⅡA and thus was designated TⅡA synthase (SmTⅡAS). Furthermore, SmTⅡAS showed a strict substrate specificity, and repression of SmTⅡAS expression in hairy root by RNAi led to increased accumulation of total dihydrofuran-tanshinones and decreased production of furan-tanshinones. We conclude that SmTⅡAS controls the metabolite flux from dihydrofuran- to furan-tanshinones, which influences medicinal properties of S. miltiorrhiza.

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Glandular trichomes (GTs) are epidermal structures that provide the first line of chemical defense against arthropod herbivores and other biotic threats. The most conspicuous structure on leaves of cultivated tomato (Solanum lycopersicum) is the type-VI GT (tVI-GT), which accumulates both flavonoids and volatile terpenoids. Although these classes of specialized metabolites are derived from distinct metabolic pathways, previous studies with a chalcone isomerase 1 (CHI1)-deficient mutant called anthocyanin free (af) showed that flavonoids are required for terpenoid accumulation in tVI-GTs. Here, we combined global transcriptomic and proteomic analyses of isolated trichomes as a starting point to show that the lack of CHI1 is associated with reduced levels of terpenoid biosynthetic transcripts and enzymes. The flavonoid deficiency in af trichomes also resulted in the upregulation of abiotic stress-responsive genes associated with DNA damage and repair. Several lines of biochemical and genetic evidence indicate that the terpenoid defect in af mutants is specific for the tVI-GT and is associated with the absence of bulk flavonoids rather than loss of CHI1 per se. A newly developed genome-scale model of metabolism in tomato tVI-GTs helped identify metabolic imbalances caused by the loss of flavonoid production. We provide evidence that flavonoid deficiency in this cell type leads to increased production of reactive oxygen species (ROS), which may impair terpenoid biosynthesis. Collectively, our findings support a role for flavonoids as ROS-scavenging antioxidants in GTs.

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes.

Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.

Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and ammonium-deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the nitrogen-deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin (tZ), a cytokinin, was upregulated in response to the nitrogen supply, but tZ appeared not to target the compensatory regulation. Our results also implied that the O. longistaminata putative ortholog of rice (Oryza sativa) C-terminally encoded peptide1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory upregulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies.

Stomatal movement is essential for plants to optimize transpiration and therefore photosynthesis. Rapid changes in the stomatal aperture are accompanied by adjustment of vacuole volume and morphology in guard cells (GCs). In Arabidopsis (Arabidopsis thaliana) leaf epidermis, stomatal development undergoes a cell-fate transition including four stomatal lineage cells: meristemoid, guard mother cell, young GC, and GC. Little is known about the mechanism underlying vacuole dynamics and vacuole formation during stomatal development. Here, we utilized whole-cell electron tomography (ET) analysis to elucidate vacuole morphology, formation, and development in different stages of stomatal lineage cells at nanometer resolution. The whole-cell ET models demonstrated that large vacuoles were generated from small vacuole stepwise fusion/maturation along stomatal development stages. Further ET analyses verified the existence of swollen intraluminal vesicles inside distinct vacuoles at certain developmental stages of stomatal lineage cells, implying a role of multivesicular body fusion in stomatal vacuole formation. Collectively, our findings demonstrate a mechanism mediating vacuole formation in Arabidopsis stomatal development and may shed light on the role of vacuoles in stomatal movement.

Histone deacetylase enzymes participate in the regulation of many aspects of plant development. However, the genome-level targets of histone deacetylation during apple (Malus domestica) fruit development have not been resolved in detail, and the mechanisms of regulation of such a process are unknown. We previously showed that the complex of ethylene response factor 4 (MdERF4) and the TOPLESS co-repressor (MdTPL4; MdERF4-MdTPL4) is constitutively active during apple fruit development (Hu et al., 2020), but whether this transcriptional repression complex is coupled to chromatin modification is unknown. Here, we show that a histone deacetylase (MdHDA19) is recruited to the MdERF4-MdTPL4 complex, thereby impacting fruit ethylene biosynthesis. Transient suppression of MdHDA19 expression promoted fruit ripening and ethylene production. To identify potential downstream target genes regulated by MdHDA19, we conducted chromatin immunoprecipitation (ChIP) sequencing of H3K9 and ChIP-quantitative polymerase chain reaction assays. We found that MdHDA19 affects ethylene production by facilitating H3K9 deacetylation and forms a complex with MdERF4-MdTPL4 to directly repress MdACS3a expression by decreasing the degree of acetylation. We demonstrate that an early-maturing-specific acetylation H3K9ac peak in MdACS3a and expression of MdACS3a were specifically up-regulated in fruit of an early-maturing, but not a late-maturing, cultivar. We provide evidence that a C-to-G mutation in the ethylene-responsive element binding factor-associated amphiphilic repression motif of MdERF4 reduces the repression of MdACS3a by the MdERF4-MdTPL4-MdHDA19 complex. Taken together, our results reveal that the MdERF4-MdTPL-MdHDA19 repressor complex participates in the epigenetic regulation of apple fruit ripening.

Plants are frequently subjected to different combinations of abiotic stresses, such as high light (HL) intensity, and elevated temperatures. These environmental conditions pose a threat to agriculture production, affecting photosynthesis, and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources toward stress responses, and suppressing growth. Here we show that the primary metabolic response of Arabidopsis (Arabidopsis thaliana) plants to HL or heat stress (HS) is different from that of plants subjected to a combination of HL and HS (HL+HS). We further demonstrate that the combined stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids coupled with decreased levels of metabolites participating in the tricarboxylic acid cycle. Among the amino acids exclusively accumulated during HL+HS, we identified the nonproteinogenic amino acid γ-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis (GLUTAMATE DESCARBOXYLASE 3 [gad3]) as well as mutants impaired in autophagy (autophagy-related proteins 5 and 9 [atg5 and atg9]), revealed that GABA plays a key role in the acclimation of plants to HL+HS, potentially by promoting autophagy. Taken together, our findings identify a role for GABA in regulating plant responses to combined stress.

Comment in doi: 10.1093/plphys/kiac079.

The common ancestor of seed plants and mosses contained homo-oligomeric cellulose synthesis complexes (CSCs) composed of identical subunits encoded by a single CELLULOSE SYNTHASE (CESA) gene. Seed plants use different CESA isoforms for primary and secondary cell wall deposition. Both primary and secondary CESAs form hetero-oligomeric CSCs that assemble and function in planta only when all the required isoforms are present. The moss Physcomitrium (Physcomitrella) patens has seven CESA genes that can be grouped into two functionally and phylogenetically distinct classes. Previously, we showed that PpCESA3 and/or PpCESA8 (class A) together with PpCESA6 and/or PpCESA7 (class B) form obligate hetero-oligomeric complexes required for normal secondary cell wall deposition. Here, we show that gametophore morphogenesis requires a member of class A, PpCESA5, and is sustained in the absence of other PpCESA isoforms. PpCESA5 also differs from the other class A PpCESAs as it is able to self-interact and does not co-immunoprecipitate with other PpCESA isoforms. These results are consistent with the hypothesis that homo-oligomeric CSCs containing only PpCESA5 subunits synthesize cellulose required for gametophore morphogenesis. Analysis of mutant phenotypes also revealed that, like secondary cell wall deposition, normal protonemal tip growth requires class B isoforms (PpCESA4 or PpCESA10), along with a class A partner (PpCESA3, PpCESA5, or PpCESA8). Thus, P. patens contains both homo-oligomeric and hetero-oligomeric CSCs.

Lignin is a complex phenylpropanoid polymer deposited in the secondary cell walls of vascular plants. Unlike most gymnosperm and eudicot lignins that are generated via the polymerization of monolignols, grass lignins additionally incorporate the flavonoid tricin as a natural lignin monomer. The biosynthesis and functions of tricin-integrated lignin (tricin-lignin) in grass cell walls and its effects on the utility of grass biomass remain largely unknown. We herein report a comparative analysis of rice (Oryza sativa) mutants deficient in the early flavonoid biosynthetic genes encoding CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), and CHI-LIKE (CHIL), with an emphasis on the analyses of disrupted tricin-lignin formation and the concurrent changes in lignin profiles and cell wall digestibility. All examined CHS-, CHI-, and CHIL-deficient rice mutants were largely depleted of extractable flavones, including tricin, and nearly devoid of tricin-lignin in the cell walls, supporting the crucial roles of CHS and CHI as committed enzymes and CHIL as a noncatalytic enhancer in the conserved biosynthetic pathway leading to flavone and tricin-lignin formation. In-depth cell wall structural analyses further indicated that lignin content and composition, including the monolignol-derived units, were differentially altered in the mutants. However, regardless of the extent of the lignin alterations, cell wall saccharification efficiencies of all tested rice mutants were similar to that of the wild-type controls. Together with earlier studies on other tricin-depleted grass mutant and transgenic plants, our results reflect the complexity in the metabolic consequences of tricin pathway perturbations and the relationships between lignin profiles and cell wall properties.

To overcome pathogen infection, plants deploy a highly efficient innate immune system, which often uses hydrogen peroxide (H2O2), a versatile reactive oxygen species, to activate downstream defense responses. H2O2 is a potential substrate of aquaporins (AQPs), the membrane channels that facilitate the transport of small compounds across plasma membranes or organelle membranes. To date, however, the functional relationship between AQPs and H2O2 in plant immunity is largely undissected. Here, we report that the rice (Oryza sativa) AQP OsPIP2;2 transports pathogen-induced apoplastic H2O2 into the cytoplasm to intensify rice resistance against various pathogens. OsPIP2;2-transported H2O2 is required for microbial molecular pattern flg22 to activate the MAPK cascade and to induce the downstream defense responses. In response to flg22, OsPIP2;2 is phosphorylated at the serine residue S125, and therefore gains the ability to transport H2O2. Phosphorylated OsPIP2;2 also triggers the translocation of OsmaMYB, a membrane-anchored MYB transcription factor, into the plant cell nucleus to impart flg22-induced defense responses against pathogen infection. On the contrary, if OsPIP2;2 is not phosphorylated, OsmaMYB remains associated with the plasma membrane, and plant defense responses are no longer induced. These results suggest that OsPIP2;2 positively regulates plant innate immunity by mediating H2O2 transport into the plant cell and mediating the translocation of OsmaMYB from plasma membrane to nucleus.

Deposition of H2A.Z and H4 acetylation by SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) and Nucleosome Acetyltransferase of H4 (NuA4) complexes in specific regulatory regions modulates transcription and development. However, little is known about these complexes in Oryza sativa (rice) development. Here, we reported that OsYAF9 and OsSWC4, two subunits of SWR1 and NuA4 complexes, are involved in rice vegetative and reproductive development. Loss of OsYAF9 resulted in reduced height, fewer tillers, fewer pollen grains, and defects in embryogenesis and seed filling. OsYAF9 directly interacted with OsSWC4 in vitro and in vivo. Loss of OsSWC4 function exhibited defects in pollen germination and failure to generate seeds, whereas knockdown of OsSWC4 resulted in reduced height and fewer tillers. The reduced height caused by OsYAF9 mutation and OsSWC4 knockdown was due to shorter internodes and defects in cell elongation, and this phenotype was rescued with gibberellin (GA) treatment, suggesting that both OsYAF9 and OsSWC4 are involved in the GA biosynthesis pathway. OsSWC4 was directly bound to the AT-rich region of GA biosynthesis genes, which in turn accomplished H2A.Z deposition and H4 acetylation at the GA biosynthesis genes with OsYAF9. Together, our study provides insights into the mechanisms involving OsSWC4 and OsYAF9 forming a protein complex to promote rice internode elongation with H2A.Z deposition and H4 acetylation.

Primary thickening determines bamboo yield and wood property. However, little is known about the regulatory networks involved in this process. This study identified a total of 58,652 genes and 150 miRNAs via transcriptome and small RNA sequencing using the underground thickening shoot samples of wild-type (WT) Moso bamboo (Phyllostachys edulis) and a thick wall (TW) variant (P. edulis "Pachyloen") at five developmental stages (WTS1/TWS1-WTS5/TWS5). A total of 14,029 (65.17%) differentially expressed genes and 68 (45.33%) differentially expressed miRNAs were identified from the WT, TW, and WTTW groups. The first two groups were composed of four pairwise combinations, each between two successive stages (WTS2/TWS2_versus_WTS1/TWS1, WTS3/TWS3_versus_WTS2/TWS2, WTS4/TWS4_versus_WTS3/TWS3, and WTS5/TWS5_versus_WTS4/TWS4), and the WTTW group was composed of five combinations, each between two relative stages (TWS1-5_versus_WTS1-5). Additionally, among the phytohormones, zeatin showed more remarkable changes in concentrations than indole-3-acetic acid, gibberellic acid, and abscisic acid throughout the five stages in the WT and the TW groups. Moreover, 125 cleavage sites were identified for 387 miRNA-mRNA pairs via degradome sequencing (P < 0.05). The dual-luciferase reporter assay confirmed that 13 miRNAs bound to 12 targets. Fluorescence in situ hybridization localized miR166 and miR160 in the shoot apical meristem and the procambium of Moso bamboo shoots at the S1 stage. Thus, primary thickening is a complex process regulated by miRNA-gene-phytohormone networks, and the miRNAome and transcriptome dynamics regulate phenotypic plasticity. These findings provide insights into the molecular mechanisms underlying wood formation and properties and propose targets for bamboo breeding.

The Yang cycle is involved in many essential metabolic pathways in plant growth and development. As extended products of the Yang cycle, the function and regulation network of ethylene and polyamines are well characterized. Nicotianamine (NA) is also a product of this cycle and works as a key metal chelator for iron (Fe) homeostasis in plants. However, interactions between the Yang cycle and NA biosynthesis remain unclear. Here, we cloned maize interveinal chlorosis 1 (mic1), encoding a 5'-methylthioadenosine nucleosidase (MTN), that is essential for 5'-methylthioadenosine (MTA) salvage and NA biosynthesis in maize (Zea mays). A single base G-A transition in the fourth exon of mic1 causes a Gly to Asp change, resulting in increased MTA, reduced Fe distribution, and growth retardation of seedlings. Knockout of ZmMIC1 but not its paralog ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis, indicating ZmMIC1 is mainly responsible for MTN activity in maize. Transcriptome analysis showed a typical response of Fe deficiency. However, metabolic analysis revealed dramatically reduced NA content in mic1, suggesting NA biosynthesis was impaired in the mutant. Exogenous application of NA transiently reversed the interveinal chlorosis phenotype of mic1 seedlings. Moreover, the mic1 mutant overexpressing a NA synthase gene not only recovered from interveinal chlorosis and growth retardation but was also fertile. These findings provide a link between the Yang cycle and NA biosynthesis, which highlights an aspect of Fe homeostasis regulation in maize.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv. Coker 312) target lines suitable for Mycobacteriophage Bxb1 recombinase-mediated gene stacking. Obtained through the empirical screening of random insertion events, each of these target lines contains a single intact copy of the target construct with precise sequences of RS2, lox, and attP sites that is not inserted within or close to a known gene or near a centromere and shows good expression of the reporter gene gfp. Gene stacking was tested with insertion of different combinations of three candidate genes for resistance to verticillium wilt into three cotton target lines: CTS1, CTS3, and CTS4. Nine site-specific integration events were recovered from 95 independently transformed embryogenic calluses. Southern and DNA sequence analyses of regenerated plants confirmed precise site-specific integration, and resistance to verticillium wilt was observed for plant CTS1i3, which has a single precise copy of site-specifically integrated DNA. These cotton target lines can serve as foundation lines for recombinase-mediated gene stacking to facilitate precise DNA integration and introgression to field cultivars.

Accurate germplasm characterization is a vital step for accelerating crop genetic improvement, which remains largely infeasible for crops such as bread wheat (Triticum aestivum L.), which has a complex genome that undergoes frequent introgression and contains many structural variations. Here, we propose a genomic strategy called ggComp, which integrates resequencing data with copy number variations and stratified single-nucleotide polymorphism densities to enable unsupervised identification of pairwise germplasm resource-based Identity-By-Descent (gIBD) blocks. The reliability of ggComp was verified in wheat cultivar Nongda5181 by dissecting parental-descent patterns represented by inherited genomic blocks. With gIBD blocks identified among 212 wheat accessions, we constructed a multi-scale genomic-based germplasm network. At the whole-genome level, the network helps to clarify pedigree relationship, demonstrate genetic flow, and identify key founder lines. At the chromosome level, we were able to trace the utilization of 1RS introgression in modern wheat breeding by hitchhiked segments. At the single block scale, the dissected germplasm-based haplotypes nicely matched with previously identified alleles of "Green Revolution" genes and can guide allele mining and dissect the trajectory of beneficial alleles in wheat breeding. Our work presents a model-based framework for precisely evaluating germplasm resources with genomic data. A database, WheatCompDB (http://wheat.cau.edu.cn/WheatCompDB/), is available for researchers to exploit the identified gIBDs with a multi-scale network.

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process.

Brassinosteroids (BRs) are plant steroids that have growth-promoting capacities, which are partly enabled by an ability to induce biosynthesis of gibberellins (GAs), a second class of plant hormones. In addition, BRs can also activate GA catabolism; here we show that in Arabidopsis (Arabidopsis thaliana) the basic helix-loop-helix transcription factor CESTA (CES) and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to this activity. CES and the BEEs are BR-regulated at the transcriptional and posttranslational level and participate in different physiological processes, including vegetative and reproduction development, shade avoidance, and cold stress responses. We show that CES/BEEs can induce the expression of the class III GA 2-oxidase GA2ox7 and that this activity is increased by BRs. In BR signaling - and CES/BEE-deficient mutants, GA2ox7 expression decreased, yielding reduced levels of GA110, a product of GA2ox7 activity. In plants that over-express CES, GA2ox7 expression is hyper-responsive to BR, GA110 levels are elevated and amounts of bioactive GA are reduced. We provide evidence that CES directly binds to the GA2ox7 promoter and is activated by BRs, but can also act by BR-independent means. Based on these results, we propose a model for CES activity in GA catabolism where CES can be recruited for GA2ox7 induction not only by BR, but also by other factors.

Tools for sequence-specific DNA binding have opened the door to new approaches in investigating fundamental questions in biology and crop development. While there are several platforms to choose from, many of the recent advances in sequence-specific targeting tools are focused on developing Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated (CRISPR-Cas)-based systems. Using a catalytically inactive Cas protein (dCas), this system can act as a vector for different modular catalytic domains (effector domains) to control a gene's expression or alter epigenetic marks such as DNA methylation. Recent trends in developing CRISPR-dCas systems include creating versions that can target multiple copies of effector domains to a single site, targeting epigenetic changes that, in some cases, can be inherited to the next generation in the absence of the targeting construct, and combining effector domains and targeting strategies to create synergies that increase the functionality or efficiency of the system. This review summarizes and compares DNA targeting technologies, the effector domains used to target transcriptional control and epi-mutagenesis, and the different CRISPR-dCas systems used in plants.

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, β, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Since its first appearance, CRISPR-Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR-Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR-Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR-Cas systems have been reported. Among them, several CRISPR-Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR-Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR-Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR-Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR-Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR-Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR-Cas tools are helping expand our plant genome engineering toolbox.

The content of organic acids greatly influences the taste and storage life of fleshy fruit. Our current understanding of the molecular mechanism of organic acid accumulation in apple (Malus domestica) fruit focuses on the aluminum-activated malate transporter 9/Ma1 gene. In this study, we identified a candidate gene, MdWRKY126, for controlling fruit acidity independent of Ma1 using homozygous recessive mutants of Ma1, namely Belle de Boskoop "BSKP" and Aifeng "AF." Analyses of transgenic apple calli and flesh and tomato (Solanum lycopersicum) fruit demonstrated that MdWRKY126 was substantially associated with malate content. MdWRKY126 was directly bound to the promoter of the cytoplasmic NAD-dependent malate dehydrogenase MdMDH5 and promoted its expression, thereby enhancing the malate content of apple fruit. In MdWRKY126 overexpressing calli, the mRNA levels of malate-associated transporters and proton pump genes also significantly increased, which contributed to the transport of malate accumulated in the cytoplasm to the vacuole. These findings demonstrated that MdWRKY126 regulates malate anabolism in the cytoplasm and coordinates the transport between cytoplasm and vacuole to regulate malate accumulation. Our study provides useful information to improve our understanding of the complex mechanism regulating apple fruit acidity.

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

Light affects many physiological and developmental processes of plants by regulating the expression and activity of light-responsive proteins. Among them, phytochrome interacting factors (PIFs) play pivotal roles in the regulation of anthocyanin accumulation and hypocotyl growth. However, the molecular mechanism is not well understood, especially in woody plants, such as apple (Malus × domestica). In this study, we identified a light-responsive PIF protein, MdPIF7, in apple and investigated the molecular mechanism of its regulation of anthocyanin biosynthesis and hypocotyl growth. We found that overexpression of MdPIF7 decreased anthocyanin accumulation in transgenic apple materials and promoted hypocotyl elongation in ectopically expressed Arabidopsis (Arabidopsis thaliana). Further investigation showed that MdPIF7 functioned by interacting with B-box 23 (MdBBX23), a positive regulator of anthocyanin biosynthesis in apple and hypocotyl growth inhibition in ectopically expressed Arabidopsis, and attenuating the transcriptional activation of MdBBX23 on LONG HYPOCOTYL 5 (MdHY5). In addition, MdPIF7 interacted with basic region leucine zipper 44 (MdbZIP44) and ethylene response factor 38 (MdERF38), two positive regulators of anthocyanin biosynthesis, and it negatively regulated MdbZIP44- and MdERF38-promoted anthocyanin accumulation by interfering with the interaction between MdbZIP44/MdERF38 and MdMYB1. Taken together, our results reveal that MdPIF7 regulates anthocyanin biosynthesis in apple and hypocotyl growth in ectopically expressed Arabidopsis through MdPIF7-MdBBX23-MdHY5 and MdPIF7-MdbZIP44/MdERF38-MdMYB1 modules. Our findings enrich the functional studies of PIF proteins and provide insights into the molecular mechanism of PIF-mediated anthocyanin biosynthesis and hypocotyl growth.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (single-nucleotide polymorphisms) that require precision genome editing tools for correction of the sequences. Although homology-directed repair of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.

Comment in doi: 10.1093/plphys/kiab602.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA. Although the role of m6A has been demonstrated in many biological processes, including embryonic development, flowering time control, microspore generation, fruit ripening, and stress responses, its contribution to other aspects of plant development still needs to be explored. Herein, we show the potential link between m6A deposition and the expansion of tomato (Solanum lycopersicum) fruits through parallel m6A-immunoprecipitation-sequencing (m6A-seq) and RNA-seq analyses. We found that global m6A levels increased during tomato fruit expansion from immature green to mature green stage. m6A-seq revealed that thousands of protein-coding genes are m6A-modified mainly in the 3'-untranslated regions. m6A-seq and RNA-seq analyses showed a positive association between m6A methylation and mRNA abundance. In particular, a large number of fruit expansion-related genes involved in hormone responses and endoreduplication were m6A modified and expressed more actively than the non-m6A-modified genes, suggesting a potential role of m6A modification in tomato fruit expansion. Importantly, altering m6A levels by direct injection of 3-deazaneplanocin A (DA; m6A writer inhibitor) or meclofenamic acid (MA; m6A eraser inhibitor) into tomato fruits suppressed fruit expansion; however, injection of exogenous DA or MA accelerated or delayed fruit ripening, respectively. Collectively, these results suggest a dynamic role of m6A methylation in the expansion and ripening of tomato fruits.

During crop cultivation, water-deficit conditions retard growth, thus reducing crop productivity. Therefore, uncovering the mechanisms behind drought tolerance is a critical task for crop improvement. Here, we show that the rice (Oryza sativa) WRKY transcription factor OsWRKY5 negatively regulates drought tolerance. We determined that OsWRKY5 was mainly expressed in developing leaves at the seedling and heading stages, and that its expression was reduced by drought stress and by treatment with NaCl, mannitol, and abscisic acid (ABA). Notably, the genome-edited loss-of-function alleles oswrky5-2 and oswrky5-3 conferred enhanced drought tolerance, measured as plant growth under water-deficit conditions. Conversely, the overexpression of OsWRKY5 in the activation-tagged line oswrky5-D resulted in higher susceptibility under the same conditions. The loss of OsWRKY5 activity increased sensitivity to ABA, thus promoting ABA-dependent stomatal closure. Transcriptome deep sequencing and reverse transcription quantitative polymerase chain reaction analyses demonstrated that the expression of abiotic stress-related genes including rice MYB2 (OsMYB2) was upregulated in oswrky5 knockout mutants and downregulated in oswrky5-D mutants. Moreover, dual-luciferase, yeast one-hybrid, and chromatin immunoprecipitation assays showed that OsWRKY5 directly binds to the W-box sequences in the promoter region of OsMYB2 and represses OsMYB2 expression, thus downregulating genes downstream of OsMYB2 in the ABA signaling pathways. Our results demonstrate that OsWRKY5 functions as a negative regulator of ABA-induced drought stress tolerance, strongly suggesting that inactivation of OsWRKY5 or manipulation of key OsWRKY5 targets could be useful to improve drought tolerance in rice cultivars.

Inorganic phosphate (Pi) is the predominant form of phosphorus (P) readily accessible to plants, and Pi Transporter 1 (PHT1) genes are the major contributors to root Pi uptake. However, the mechanisms underlying the transport and recycling of Pi within plants, which are vital for optimizing P use efficiency, remain elusive. Here, we characterized a functionally unknown rice (Oryza sativa) PHT1 member barely expressed in roots, OsPHT1;7. Yeast complementation and Xenopus laevis oocyte assay demonstrated that OsPHT1;7 could mediate Pi transport. Reverse-transcription quantitative polymerase chain reaction and histochemical analyses showed that OsPHT1;7 was preferentially expressed in source leaves and nodes. A further fine-localization analysis by immunostaining showed that OsPHT1;7 expression was restricted in the vascular bundle (VB) sheath and phloem of source leaves as well as in the phloem of regular/diffuse- and enlarged-VBs of nodes. In accordance with this expression pattern, mutation of OsPHT1;7 led to increased and decreased P distribution in source (old leaves) and sink organs (new leaves/panicles), respectively, indicating that OsPHT1;7 is involved in P redistribution. Furthermore, OsPHT1;7 showed an overwhelmingly higher transcript abundance in anthers than other PHT1 members, and ospht1;7 mutants were impaired in P accumulation in anthers but not in pistils or husks. Moreover, the germination of pollen grains was significantly inhibited upon OsPHT1;7 mutation, leading to a >80% decrease in seed-setting rate and grain yield. Taken together, our results provide evidence that OsPHT1;7 is a crucial Pi transporter for Pi transport and recycling within rice plants, stimulating both vegetative and reproductive growth.

As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca2+ signature is hampered by limited tools for simultaneously monitoring dynamic Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca2+ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca2+ and apical plasma membrane Ca2+ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca2+ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca2+ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca2+ signatures in plants.

Comment in doi: 10.1093/plphys/kiac061.

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein 11/S locus cysteine-rich protein and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we detected the presence of autophagic bodies with self-incompatible (SI) pollinations in Arabidopsis lyrata and transgenic Arabidopsis thaliana lines, but whether autophagy was essential for self-pollen rejection was unknown. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) and ATG5 genes into two different transgenic SI A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and Arabidopsis halleri self-incompatibility genes, we demonstrated that disrupting autophagy weakened their SI responses in the stigma. When the atg7 or atg5 mutations were present, an increased number of SI pollen was found to hydrate and form pollen tubes that successfully fertilized the SI pistils. Additionally, we confirmed the presence of GFP-ATG8a-labeled autophagosomes in the stigmatic papillae following SI pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the intracellular mechanisms employed in the transgenic A. thaliana stigmas to reject self-pollen.

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs) indispensable for chloroplast biogenesis. Salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce the expression of PhANGs, leading to photoinhibition of photosystem II and singlet oxygen (1O2) burst in chloroplasts. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1; 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. This earlier finding has initiated research on the potential role of GLK1/2 and EX1 in SA signaling. Consistent with this view, we reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities in Arabidopsis (Arabidopsis thaliana). Overexpression of LSD1 repressed GLK target genes, including PhANGs, whereas loss of LSD1 enhanced their expression. Remarkably, LSD1 overexpression inhibited chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. Subsequent chromatin immunoprecipitation coupled with expression analyses further revealed that LSD1 inhibits the DNA-binding activity of GLK1 toward its target promoters. SA-induced nuclear-targeted SIB1 proteins appeared to interrupt the LSD1-GLK interaction, and the subsequent SIB1-GLK interaction activated EX1-mediated 1O2 signaling, elucidating antagonistic modules SIB1 and LSD1 in the regulation of GLK activity. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily. Moreover, suicide genes have been used to trigger self-elimination of transgenic plants, greatly improving the efficiency of isolating the desired transgene-free plants. Transgenes can also be excised from plant genomes using site-specific recombination, transposition or gene editing nucleases, providing a strategy for editing asexually produced plants. Finally, haploid induction coupled with gene editing may make it feasible to edit plants that are recalcitrant to transformation. Here, we evaluate the strengths and weaknesses of recently developed approaches for obtaining edited plants without transgene residuals.

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP+. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI-LHCI-LHCII supercomplex. The binding site(s) of the "additional" LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that "additional" LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR-Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR-Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.

Growth promotion induced by the endosymbiont Piriformospora indica has been observed in various plants; however, except growth phytohormones, specific functional metabolites involved in P. indica-mediated growth promotion are unknown. Here, we used a gas chromatography-mass spectrometry-based untargeted metabolite analysis to identify tomato (Solanum lycopersicum) metabolites whose levels were altered during P. indica-mediated growth promotion. Metabolomic multivariate analysis revealed several primary metabolites with altered levels, with putrescine (Put) induced most significantly in roots during the interaction. Further, our results indicated that P. indica modulates the arginine decarboxylase (ADC)-mediated Put biosynthesis pathway via induction of SlADC1 in tomato. Piriformospora indica did not promote growth in Sladc1-(virus-induced gene silencing of SlADC1) lines of tomato and showed less colonization. Furthermore, using LC-MS/MS we showed that Put promoted growth by elevation of auxin (indole-3-acetic acid) and gibberellin (GA4 and GA7) levels in tomato. In Arabidopsis (Arabidopsis thaliana) adc knockout mutants, P. indica colonization also decreased and showed no plant growth promotion, and this response was rescued upon exogenous application of Put. Put is also important for hyphal growth of P. indica, indicating that it is co-adapted by both host and microbe. Taken together, we conclude that Put is an essential metabolite and its biosynthesis in plants is crucial for P. indica-mediated plant growth promotion and fungal growth.