Hydroxyproline O-arabinosyltransferase Research Articles

GmNLP1 and GmNLP4 activate nitrate-induced CLE peptides NIC1a/b to mediate nitrate-regulated root nodulation.

Symbiotic nitrogen fixation is an energy-intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR-Cas9-mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate-tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate-triggered inhibition of nodulation, as the expression of nitrate-responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O-arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4-GmNIC1a/b module in mediating high nitrate-induced inhibition of nodulation.

Tomato arabinosyltransferase prevents precocious senescence

Senescence, a crucial developmental process in the life cycle of plants, involves programmed destruction of cellular components of leaves. The onset of senescence is synchronized with other developmental processes for successful reproduction since senescence eventually leads to cell death. Arabinosyltransferase FASCIATED AND BRANCHED 2 (FAB2) is known to control meristem proliferation. Here, we show that FAB2 can inhibit premature leaf senescence in tomato plants. Both chemically mutagenized and CRISPR-generated fab2 mutants exhibited excessively accelerated senescence, which resulted in sterility. Transcriptome analysis revealed that FAB2 extended leaf longevity by suppressing transcription of genes highly expressed in mature leaves. Transcription of FAB2 was increased in younger leaves, potentially inhibiting premature leaf senescence. The precocious senescence of fab2 mutants was in contrast to fasciated inflorescence (fin) mutants, which carried mutations in a hydroxyproline O-arabinosyltransferase gene, leading to meristem overproliferation. Our observations indicate that complex genetic hierarchy in the cascade of tomato arabinosyltransferases could control different aspects of developmental processes such as stem cell proliferation and senescence.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

The role of CLAVATA signalling in the negative regulation of mycorrhizal colonization and nitrogen response of tomato.

Plants form mutualistic nutrient-acquiring symbioses with microbes, including arbuscular mycorrhizal fungi. The formation of these symbioses is costly, and plants employ a negative feedback loop termed autoregulation of mycorrhizae (AOM) to limit formation of arbuscular mycorrhizae (AM). We provide evidence for the role of one leucine-rich repeat receptor-like kinase (FAB), a hydroxyproline O-arabinosyltransferase enzyme (FIN), and additional evidence for one receptor-like protein (SlCLV2) in the negative regulation of AM formation in tomato. Reciprocal grafting experiments suggest that the FAB gene acts locally in the root, while the SlCLV2 gene may act in both the root and the shoot. External nutrients including phosphate and nitrate can also strongly suppress AM formation. We found that FAB and FIN are required for nitrate suppression of AM but are not required for the powerful suppression of AM colonization by phosphate. This parallels some of the roles of legume homologues in the autoregulation of the more recently evolved symbioses with nitrogen-fixing bacteria leading to nodulation. This deep homology in the symbiotic role of these genes suggests that in addition to the early signalling events that lead to the establishment of AM and nodulation, the autoregulation pathway might also be considered part of the common symbiotic toolkit that enabled plants to form beneficial symbioses.

The role of CLV1, CLV2 and HPAT homologues in the nitrogen-regulation of root development.

Plants use a variety of signals to control root development, including in modifying root development in response to nutrient stress. For example, in response to nitrogen (N) stress, plants dramatically modulate root development, including the formation of N-fixing nodules in legumes. Recently, specific CLE peptides and/or receptors important for their perception, including CLV1 and CLV2, have been found to play roles in root development, including in response to N supply. In the legume Medicago truncatula, this response also appears to be influenced by RDN1, a member of the hydroxyproline-O-arabinosyltransferase (HPAT) family which can modify specific CLE peptides. However, it is not known if this signalling pathway plays a central role in root development across species, and in particular root responses to N. In this study, we systematically examined the role of the CLV signalling pathway genes in root development of the legume pea (Pisum sativum) and non-legume tomato (Solanum lycopersicum) using a mutant-based approach. This included a detailed examination of root development in response to N in tomato mutants disrupted in CLV1- or CLV2-like genes or HPAT family member FIN. We found no evidence for a role of these genes in pea seedling root development. Furthermore, the CLV1-like FAB gene did not influence tomato root development, including the root response to N supply. In contrast, both CLV2 and the HPAT gene FIN appear to positively influence root size in tomato but do not mediate root responses to N. These results suggest the function of these genes may vary somewhat in different species, including the N regulation of root architecture.

Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O-arabinosyltransferase mutants.

SummaryHYDROXYPROLINE O‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana, HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft‐like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed‐set phenotype, we identified a missense mutation in exo70a2, a predicted member of the vesicle‐tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst‐mediated secretion. To monitor the trafficking of Hyp‐Ara modified proteins, we generated an HPAT‐targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.

The CLE53-SUNN genetic pathway negatively regulates arbuscular mycorrhiza root colonization in Medicago truncatula.

Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O-arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53, rdn1, and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMF-colonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels.

PLENTY, a hydroxyproline O-arabinosyltransferase, negatively regulates root nodule symbiosis in Lotus japonicus.

Legumes can survive in nitrogen-deficient environments by forming root-nodule symbioses with rhizobial bacteria; however, forming nodules consumes energy, and nodule numbers must thus be strictly controlled. Previous studies identified major negative regulators of nodulation in Lotus japonicus, including the small peptides CLAVATA3/ESR (CLE)-RELATED-ROOT SIGNAL1 (CLE-RS1), CLE-RS2, and CLE-RS3, and their putative major receptor HYPERNODULATION AND ABERRANT ROOT FORMATION1 (HAR1). CLE-RS2 is known to be expressed in rhizobia-inoculated roots, and is predicted to be post-translationally arabinosylated, a modification essential for its activity. Moreover, all three CLE-RSs suppress nodulation in a HAR1-dependent manner. Here, we identified PLENTY as a gene responsible for the previously isolated hypernodulation mutant plenty. PLENTY encoded a hydroxyproline O-arabinosyltransferase orthologous to ROOT DETERMINED NODULATION1 in Medicago truncatula. PLENTY was localized to the Golgi, and an in vitro analysis of the recombinant protein demonstrated its arabinosylation activity, indicating that CLE-RS1/2/3 may be substrates for PLENTY. The constitutive expression experiments showed that CLE-RS3 was the major candidate substrate for PLENTY, suggesting the substrate preference of PLENTY for individual CLE-RS peptides. Furthermore, a genetic analysis of the plenty har1 double mutant indicated the existence of another PLENTY-dependent and HAR1-independent pathway negatively regulating nodulation.

Triarabinosylation is required for nodulation-suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum.

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.

ROOT DETERMINED NODULATION1 Is Required for M. truncatula CLE12, But Not CLE13, Peptide Signaling through the SUNN Receptor Kinase.

The combinatorial interaction of a receptor kinase and a modified CLE peptide is involved in several developmental processes in plants, including autoregulation of nodulation (AON), which allows legumes to limit the number of root nodules formed based on available nitrogen and previous rhizobial colonization. Evidence supports the modification of CLE peptides by enzymes of the hydroxyproline O-arabinosyltransferase (HPAT/RDN) family. Here, we show by grafting and genetic analysis in Medicago truncatula that, in the AON pathway, RDN1, functioning in the root, acts upstream of the receptor kinase SUNN, functioning in the shoot. As expected for a glycosyltransferase, we found that RDN1 and RDN2 proteins are localized to the Golgi, as was shown previously for AtHPAT1. Using composite plants with transgenic hairy roots, we show that RDN1 and RDN2 orthologs from dicots as well as a related RDN gene from rice (Oryza sativa) can rescue the phenotype of rdn1-2 when expressed constitutively, but the less related MtRDN3 cannot. The timing of the induction of MtCLE12 and MtCLE13 peptide genes (negative regulators of AON) in nodulating roots is not altered by the mutation of RDN1 or SUNN, although expression levels are higher. Plants with transgenic roots constitutively expressing MtCLE12 require both RDN1 and SUNN to prevent nodule formation, while plants constitutively expressing MtCLE13 require only SUNN, suggesting that the two CLEs have different requirements for function. Combined with previous work, these data support a model in which RDN1 arabinosylates MtCLE12, and this modification is necessary for the transport and/or reception of the AON signal by the SUNN kinase.

A cascade of arabinosyltransferases controls shoot meristem size in tomato

Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation.