Dynamic Nutrient Signaling Networks in Plants.

Nutrients are vital to life through intertwined sensing, signaling, and metabolic processes. Emerging research focuses on how distinct nutrient signaling networks integrate and coordinate gene expression, metabolism, growth, and survival. We review the multifaceted roles of sugars, nitrate, and phosphate as essential plant nutrients in controlling complex molecular and cellular mechanisms of dynamic signaling networks. Key advances in central sugar and energy signaling mechanisms mediated by the evolutionarily conserved master regulators HEXOKINASE1 (HXK1), TARGET OF RAPAMYCIN (TOR), and SNF1-RELATED PROTEIN KINASE1 (SNRK1) are discussed. Significant progress in primary nitrate sensing, calcium signaling, transcriptome analysis, and root-shoot communication to shape plant biomass and architecture are elaborated. Discoveries on intracellular and extracellular phosphate signaling and the intimate connections with nitrate and sugar signaling are examined. This review highlights the dynamic nutrient, energy, growth, and stress signaling networks that orchestrate systemwide transcriptional, translational, and metabolic reprogramming, modulate growth and developmental programs, and respond to environmental cues.

Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3 (refs. 6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

Primary nitrate responses mediated by calcium signalling and diverse protein phosphorylation.

Nitrate, the major source of inorganic nitrogen for plants, is a critical signal controlling nutrient transport and assimilation and adaptive growth responses throughout the plant. Understanding how plants perceive nitrate and how this perception is transduced into responses that optimize growth are important for the rational improvement of crop productivity and for mitigating pollution from the use of fertilizers. This review highlights recent findings that reveal key roles of cytosolic-nuclear calcium signalling and dynamic protein phosphorylation via diverse mechanisms in the primary nitrate response (PNR). Nitrate-triggered calcium signatures as well as the critical functions of subgroup III calcium-sensor protein kinases, a specific protein phosphatase 2C, and RNA polymerase II C-terminal domain phosphatase-like 3 are discussed. Moreover, genome-wide meta-analysis of nitrate-regulated genes encoding candidate protein kinases and phosphatases for modulating critical phosphorylation events in the PNR are elaborated. We also consider how phosphoproteomics approaches can contribute to the identification of putative regulatory protein kinases in the PNR. Exploring and integrating experimental strategies, new methodologies, and comprehensive datasets will further advance our understanding of the molecular and cellular mechanisms underlying the complex regulatory processes in the PNR.

NIN-like protein 7 transcription factor is a plant nitrate sensor.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

Transcriptomic profiling of Brassica napus responses to Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen of plants. Unlike the well-characterized plant defense responses to highly adapted bacterial phytopathogens, little is known about plant response to P. aeruginosa infection. In this study, we examined the Brassica napus (canola) tissue-specific response to P. aeruginosa infection using RNA sequencing. Transcriptomic analysis of canola seedlings over a 5 day P. aeruginosa infection revealed that many molecular processes involved in plant innate immunity were up-regulated, whereas photosynthesis was down-regulated. Phytohormones control many vital biological processes within plants, including growth and development, senescence, seed setting, fruit ripening, and innate immunity. The three main phytohormones involved in plant innate immunity are salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Many bacterial pathogens have evolved multiple strategies to manipulate these hormone responses in order to infect plants successfully. Interestingly, gene expression within all three phytohormone (SA, JA, and ET) signaling pathways was up-regulated in response to P. aeruginosa infection. This study identified a unique plant hormone response to the opportunistic bacterial pathogen P. aeruginosa infection.

Cloning and characterization of two novel lipid transfer protein I genes in Vigna radiata.

Two full-length lipid transfer protein (LTP) cDNAs were isolated from mungbean (Vigna radiata) and designated Vrltp1 and Vrltp2. The deduced amino acid sequences contain the two highly conserved pentapeptides characteristic of plant LTPIs suggesting these Vrltps belong to the LTPI gene family. Vrltp1 mRNA was detected in developing seeds, but Vrltp2 mRNA was not. Within the vegetative tissues, the Vrltp1 and Vrltp2 mRNAs were present only in leaves and stems, but not root tips. Salt and dehydration stresses and exogenous abscisic acid (ABA) treatments resulted in increased mRNA levels of both Vrltps in leaves. We suggest that these unique Vrltps are specific to growing shoot tissues, and may play an important role in plant acclimation to water stress.

AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings.

BACKGROUND: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. RESULTS: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using ß-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein-protein interactions in physiological contexts. CONCLUSIONS: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein-protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.

Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis.

Large-scale genetic screens in Arabidopsis are a powerful approach for molecular dissection of complex signaling networks. However, map-based cloning can be time-consuming or even hampered due to low chromosomal recombination. Current strategies using next generation sequencing for molecular identification of mutations require whole genome sequencing and advanced computational devises and skills, which are not readily accessible or affordable to every laboratory. We have developed a streamlined method using parallel massive sequencing for mutant identification in which only targeted regions are sequenced. This targeted parallel sequencing (TPSeq) method is more cost-effective, straightforward enough to be easily done without specialized bioinformatics expertise, and reliable for identifying multiple mutations simultaneously. Here, we demonstrate its use by identifying three novel nitrate-signaling mutants in Arabidopsis.

Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation.

To counteract fluctuating nutrient environments, plants have evolved high- and low-affinity uptake systems. These two systems were traditionally thought to be genetically distinct, but, recently, two Arabidopsis transporters, AtKUP1 and CHL1, were shown to have dual affinities. However, little is known about how a dual-affinity transporter works and the advantages of having a dual-affinity transporter. This study demonstrates that, in the case of CHL1, switching between the two modes of action is regulated by phosphorylation at threonine residue 101; when phosphorylated, CHL1 functions as a high-affinity nitrate transporter, whereas, when dephosphorylated, it functions as a low-affinity nitrate transporter. This regulatory mechanism allows plants to change rapidly between high- and low-affinity nitrate uptake, which may be critical when competing for limited nitrogen. These results demonstrate yet another regulatory role of phosphorylation in plant physiology.