VPT-like genes modulate Rhizobium-legume symbiosis and phosphorus adaptation.

Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium-legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium-legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root-to-shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long-distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 and MtVPT3 modulate phosphorus adaptation and rhizobia-legume symbiosis, possibly by regulating long-distance Pi transport.

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

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

Transport and homeostasis of potassium and phosphate: limiting factors for sustainable crop production.

Potassium (K) and phosphate (Pi) are both macronutrients essential for plant growth and crop production, but the unrenewable resources of phosphorus rock and potash have become limiting factors for food security. One critical measure to help solve this problem is to improve nutrient use efficiency (NUE) in plants by understanding and engineering genetic networks for ion uptake, translocation, and storage. Plants have evolved multiple systems to adapt to various nutrient conditions for growth and production. Within the NUE networks, transport proteins and their regulators are the primary players for maintaining nutrient homeostasis and could be utilized to engineer high NUE traits in crop plants. A large number of publications have detailed K+ and Pi transport proteins in plants over the past three decades. Meanwhile, the discovery and validation of their regulatory mechanisms are fast-track topics for research. Here, we provide an overview of K+ and Pi transport proteins and their regulatory mechanisms, which participate in the uptake, translocation, storage, and recycling of these nutrients in plants.

An ABC transporter complex encoded by Aluminum Sensitive 3 and NAP3 is required for phosphate deficiency responses in Arabidopsis.

Phosphate is essential for cell metabolism in all organisms. As it is often limiting in the soil, plants have evolved various mechanisms to cope with low-phosphate conditions. Here, we report that Aluminum Sensitive 3 and NAP3, two genes previously identified to function in aluminum tolerance, play a critical role in plant response to phosphate deficiency. Two T-DNA insertional alleles of ALS3 gene in Arabidopsis showed hypersensitive responses to phosphate limiting conditions. Compared to the wild type, als3 mutant plants exhibited more severe root growth inhibition and developed more root hairs under phosphate starvation. Interestingly, these phenotypic changes occurred only when the low-phosphate medium is supplemented with sucrose, suggesting that ALS3 regulates low-phosphate response in a sugar-dependent manner. Furthermore, NAP3, a gene encoding the nucleotide binding domain protein that physically interacts with ALS3, was implicated in the same pathway in response to low-P. The nap3 mutant showed the same phenotype as the als3 mutant when grown on phosphate depletion medium. We conclude that ALS3 and NAP3 protein form an ABC transporter complex that is required for sugar-dependent response to phosphate deficiency.

A calcium sensor-regulated protein kinase, CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, is required for pollen tube growth and polarity.

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca2+ throughout the bulging tip, whereas LaCl3, a Ca2+ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca2+ homeostasis through its potential function in the modulation of Ca2+ influx.

An endoplasmic reticulum magnesium transporter is essential for pollen development in Arabidopsis.

Magnesium is one of the essential macro-elements for plant growth and development, participated in photosynthesis and various metabolic processes. The Mg-transport abilities of the AtMGT (Magnesium Transporter) genes were identified in bacteria or yeast mutant system. In our previous studies, both the AtMGT5 and AtMGT9 were found essential for pollen development in Arabidopsis. Here we report another AtMGT member, AtMGT4, which was localized to the endoplasmic reticulum, was essential for pollen development as well. AtMGT4 expressed notably in pollen grains from bicellular pollen stage to mature pollen stage. A T-DNA insertional mutant of the gene, named mgt4-1, showed pollen abortive phenotype, thus we could not get any homozygous mutant from progenies of self-crossed +/mgt4-1 plants. Meanwhile, nearly half of pollens in AtMGT4-RNAi transgenic lines were sterile, consistent with the phenotype of +/mgt4-1 mutant. Transgenic plants expressing AtMGT4 in the mgt4-1 background could recover the pollen fertility to the wild type. Together, our findings demonstrated that the disruption of AtMGT4 in Arabidopsis could cause a defect of pollen development. The visible pollen abortion appeared at bicellular pollen stage in +/mgt4-1.

A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth.

Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neurons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code and decode Ca(2+) signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32, controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca(2+) accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activation of CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity. Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, similar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading to more severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in which calcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca(2+) oscillations in the polar growth of pollen tubes.

Alternative oxidases (AOX1a and AOX2) can functionally substitute for plastid terminal oxidase in Arabidopsis chloroplasts.

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.

Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis.

Magnesium (Mg(2+)) is abundant in plant cells and plays a critical role in many physiological processes. A 10-member gene family AtMGT (also known as AtMRS2) was identified in Arabidopsis, which belongs to a eukaryote subset of the CorA superfamily, functioning as Mg(2+) transporters. Some family members (AtMGT1 and AtMGT10) function as high-affinity Mg(2+) transporter and could complement bacterial mutant or yeast mutant lacking Mg(2+) transport capability. Here we report an AtMGT family member, AtMGT9, that functions as a low-affinity Mg(2+) transporter, and is essential for pollen development. The functional complementation assay in Salmonella mutant strain MM281 showed that AtMGT9 is capable of mediating Mg(2+) uptake in the sub-millimolar range of Mg(2+). The AtMGT9 gene was expressed most strongly in mature anthers and was also detectable in vascular tissues of the leaves, and in young roots. Disruption of AtMGT9 gene expression resulted in abortion of half of the mature pollen grains in heterozygous mutant +/mgt9, and no homozygous mutant plant was obtained in the progeny of selfed +/mgt9 plants. Transgenic plants expressing AtMGT9 in these heterozygous plants can recover the pollen phenotype to the wild type. In addition, AtMGT9 RNAi transgenic plants also showed similar abortive pollen phenotype to mutant +/mgt9. Together, our results demonstrate that AtMGT9 functions as a low-affinity Mg(2+) transporter that plays a crucial role in male gametophyte development and male fertility.

A mitochondrial magnesium transporter functions in Arabidopsis pollen development.

Magnesium is an abundant divalent cation in plant cells and plays a critical role in many physiological processes. We have previously described the identification of a 10-member Arabidopsis gene family encoding putative magnesium transport (MGT) proteins. Here, we report that a member of the MGT family, AtMGT5, functions as a dual-functional Mg-transporter that operates in a concentration-dependent manner, namely it serves as a Mg-importer at micromolar levels and facilitates the efflux in the millimolar range. The AtMGT5 protein is localized in the mitochondria, suggesting that AtMGT5 mediates Mg-trafficking between the cytosol and mitochondria. The AtMGT5 gene was exclusively expressed in anthers at early stages of flower development. Examination of two independent T-DNA insertional mutants of AtMGT5 gene demonstrated that AtMGT5 played an essential role for pollen development and male fertility. This study suggests a critical role for Mg(2+) transport between cytosol and mitochondria in male gametogenesis in plants.

A chloroplast FKBP interacts with and affects the accumulation of Rieske subunit of cytochrome bf complex.

Immunophilins are intracellular receptors of the immunosuppressants cyclosporin A, FK506, and rapamycin. Although all immunophilins possess peptidyl-prolyl isomerase activity and are identified from a wide range of organisms, little is known about their cellular functions. We report the characterization and functional analysis of an FK506 and rapamycin-binding protein (AtFKBP13) from Arabidopsis. The AtFKBP13 protein is synthesized as a precursor that is imported into chloroplasts and processed to the mature form located in the thylakoid lumen, as shown by chloroplast import assays and Western blot analysis. Experiments show that AtFKBP13 is translocated across the thylakoid membrane by the DeltapH-dependent pathway. Yeast two-hybrid screening identified Rieske FeS protein, a subunit of the cytochrome bf complex in the photosynthetic electron transport chain, as an interacting partner for AtFKBP13. Both yeast two-hybrid and in vitro protein-protein interaction assays showed that the precursor, but not the mature form, of AtFKBP13 interacted with Rieske protein, suggesting that interaction between the two proteins occurs along the import pathway. When AtFKBP13 expression was suppressed by RNA interference method, the level of Rieske protein was significantly increased in the transgenic plants.

A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis.

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene (AtPTEN1) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.