Biochemical Genetics of Metabolic Plasticity

The transition to flowering is governed by different pathways integrating endogenous and exogenous signals. Here, we evaluated the role of the phytohormone cytokinin (CK) in regulating Arabidopsis thaliana flowering time. By analyzing key mutants in CK metabolism, transport and signalling, we found that the hormone promotes flowering under both long-day (LD) and short-day (SD) conditions, with a stronger impact on flowering under SDs. Genetic analyses indicated that both trans- and cis-zeatin regulate the floral transition, while isopentenyladenine plays a minor role. Blocking CK export from roots and reciprocal grafting experiments revealed that root-derived CK is an important flowering signal. Perception and transmission of the CK flowering signal depended on distinct CK receptors, phosphotransmitter proteins and several B-type response regulators. Further, CK functioned through floral integrators such as OVEREXPRESSION OF CONSTANS1 (SOC1) and components of the age pathway. The CK status of plants affected the levels of the age pathway microRNAs miR156 and miR172. Cytokinin-promoted flowering required the miR156-target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE15 (SPL15) and miR172, and the late-flowering phenotype of LD-grown CK-deficient plants depended on miR172-targeted APETALA2 (AP2)-like genes encoding floral repressors. Collectively, this study shows that CK regulates flowering time through the two-component signaling system and components of the age pathway, providing a genetic framework for future investigations.

Herbivores sharing host plants are often temporally and spatially separated, limiting direct interactions between them. Nevertheless, as observed in numerous aboveground study systems, they can reciprocally influence each other via systemically induced plant responses. In contrast, examples of such plant-mediated interactions between belowground herbivores are scarce; however, we postulated that they similarly occur, given the large diversity of root-interacting soil organisms. To test this hypothesis, we analyzed the performance of cabbage root fly (Delia radicum) larvae feeding on the main roots of field mustard (Brassica rapa) plants whose fine roots were infected by the root-knot nematode (Meloidogyne incognita). Simultaneously, we studied the effects of M. incognita on D. radicum-induced defense responses and the accumulation of primary metabolites in the main root. We observed that almost 1.5 times as many D. radicum adults emerged from nematode-infected plants, indicating a facilitation effect of M. incognita infection. Although we observed increases in the accumulation of proteins and two essential amino acids, the strongest effect of nematode infection was visible in the defense response to D. radicum. We observed a 1.5 times higher accumulation of the defense-related phytohormone JA-Ile in response to D. radicum on nematode-infected plants, coinciding with a 75% increase in indole glucosinolate concentrations. Contrastingly, concentrations of aliphatic glucosinolates, secondary metabolites negatively affecting D. radicum, were 10-25% lower in nematode-infected plants. We hypothesize that the attenuated aliphatic glucosinolate concentrations result from antagonistic interactions between biosynthetic pathways of both glucosinolate classes, which was reflected in the expression of key biosynthesis genes. Our results provide explicit evidence of plant-mediated interactions between belowground organisms, likely via systemically induced responses in roots.

The transition to flowering is governed by different pathways integrating endogenous and exogenous signals. Here, we evaluated the role of the phytohormone cytokinin (CK) in regulating Arabidopsis thaliana flowering time. By analyzing key mutants in CK metabolism, transport and signalling, we found that the hormone promotes flowering under both long-day (LD) and short-day (SD) conditions, with a stronger impact on flowering under SDs. Genetic analyses indicated that both trans- and cis-zeatin regulate the floral transition, while isopentenyladenine plays a minor role. Blocking CK export from roots and reciprocal grafting experiments revealed that root-derived CK is an important flowering signal. Perception and transmission of the CK flowering signal depended on distinct CK receptors, phosphotransmitter proteins and several B-type response regulators. Further, CK functioned through floral integrators such as OVEREXPRESSION OF CONSTANS1 (SOC1) and components of the age pathway. The CK status of plants affected the levels of the age pathway microRNAs miR156 and miR172. Cytokinin-promoted flowering required the miR156-target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE15 (SPL15) and miR172, and the late-flowering phenotype of LD-grown CK-deficient plants depended on miR172-targeted APETALA2 (AP2)-like genes encoding floral repressors. Collectively, this study shows that CK regulates flowering time through the two-component signaling system and components of the age pathway, providing a genetic framework for future investigations.

Herbivores sharing host plants are often temporally and spatially separated, limiting direct interactions between them. Nevertheless, as observed in numerous aboveground study systems, they can reciprocally influence each other via systemically induced plant responses. In contrast, examples of such plant-mediated interactions between belowground herbivores are scarce; however, we postulated that they similarly occur, given the large diversity of root-interacting soil organisms. To test this hypothesis, we analyzed the performance of cabbage root fly (Delia radicum) larvae feeding on the main roots of field mustard (Brassica rapa) plants whose fine roots were infected by the root-knot nematode (Meloidogyne incognita). Simultaneously, we studied the effects of M. incognita on D. radicum-induced defense responses and the accumulation of primary metabolites in the main root. We observed that almost 1.5 times as many D. radicum adults emerged from nematode-infected plants, indicating a facilitation effect of M. incognita infection. Although we observed increases in the accumulation of proteins and two essential amino acids, the strongest effect of nematode infection was visible in the defense response to D. radicum. We observed a 1.5 times higher accumulation of the defense-related phytohormone JA-Ile in response to D. radicum on nematode-infected plants, coinciding with a 75% increase in indole glucosinolate concentrations. Contrastingly, concentrations of aliphatic glucosinolates, secondary metabolites negatively affecting D. radicum, were 10-25% lower in nematode-infected plants. We hypothesize that the attenuated aliphatic glucosinolate concentrations result from antagonistic interactions between biosynthetic pathways of both glucosinolate classes, which was reflected in the expression of key biosynthesis genes. Our results provide explicit evidence of plant-mediated interactions between belowground organisms, likely via systemically induced responses in roots.

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defense versus growth’ responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1 (COI1)-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA-amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 (GH3) and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1 (ILR1)/ILR1-like (ILL) families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se.

Small-molecule phytohormones exert control over plant growth, development, and stress responses by coordinating the patterns of gene expression within and between cells. Increasing evidence indicates that currently recognized plant hormones are part of a larger group of regulatory metabolites that have acquired signaling properties during the evolution of land plants. This rich assortment of chemical signals reflects the tremendous diversity of plant secondary metabolism, which offers evolutionary solutions to the daunting challenges of sessility and other unique aspects of plant biology. A major gap in our current understanding of plant regulatory metabolites is the lack of insight into the direct targets of these compounds. Here, we illustrate the blurred distinction between classical phytohormones and other bioactive metabolites by highlighting the major scientific advances that transformed the view of jasmonate from an interesting floral scent to a potent transcriptional regulator. Lessons from jasmonate research generally apply to other phytohormones and thus may help provide a broad understanding of regulatory metabolite–protein interactions. In providing a framework that links small-molecule diversity to transcriptional plasticity, we hope to stimulate future research to explore the evolution, functions, and mechanisms of perception of a broad range of plant regulatory metabolites.

In Nicotiana benthamiana, the expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) triggers RECOGNITION OF XOPQ1 (ROQ1)-dependent effector-triggered immunity (ETI) responses accompanied by the accumulation of plastids around the nucleus and the formation of stromules. Both plastid clustering and stromules were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here, we utilized transient expression experiments to determine whether XopQ-triggered plastid reactions are a result of XopQ perception by the immune receptor ROQ1 or a consequence of XopQ virulence activity. We found that N. benthamiana mutants lacking ROQ1, ENHANCED DISEASE SUSCEPTIBILITY 1, or the helper NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT IMMUNE RECEPTORS (NLRs) N-REQUIRED GENE 1 (NRG1) and ACTIVATED DISEASE RESISTANCE GENE 1 (ADR1), fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the nrg1_adr1 double mutant. This analysis aligns XopQ-triggered stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response and that both NRG1 and ADR1 hNLRs play a role in this ETI response.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root’s response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate–sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.