Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.     

Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana

Cytokinin signaling in Arabidopsis thaliana utilizes a multi-step two-component signaling (TCS) system comprised of sensor histidine kinases (AHKs), histidine phosphotransfer proteins (AHPs), and response regulators (ARRs). Recent studies have suggested that the cytokinin TCS system is involved in a variety of other signaling and metabolic pathways. To further explore a potential function of the cytokinin TCS in the Arabidopsis dehydration stress response, we investigated the expression of all type-A ARR genes and a type-C ARR, ARR22, in both wild type and ahk single, double, and triple mutants in response to dehydration compared to cytokinin as well as dehydration tolerance of ahk mutants. We found that drought significantly induced the expression of a subset of ARR genes, ARR5, ARR7, ARR15, and ARR22. The results of expression analyses in ahk single, double, and triple mutants demonstrated that the cytokinin receptors AHK2 and AHK3 are redundantly involved in dehydration-inducible expression of ARR7, but not that of ARR5, ARR15, or ARR22. Dehydration tolerance assays showed that ahk2 and ahk3 single mutants exhibited enhanced dehydration tolerance compared with that of wild-type plants and ahk4 mutants, and that ahk2 ahk3 double mutants exhibited stronger drought tolerance than that of ahk3 ahk4, which exhibited more enhanced drought tolerance than that of wild-type plants and ahk single mutants. Taken together, these results demonstrate that while the cytokinin receptors AHK2 and AHK3 are critically involved in the dehydration tolerance response, both cytokinin receptor-dependent pathway and receptor-independent pathway occur in the dehydration response regulating ARR gene expression. In addition, preincubating ahk2, ahk3, ahk4, and the wild-type plants with cytokinin induced enhanced dehydration stress tolerance in these plants, demonstrating that cytokinins are involved in regulating plant response to dehydration stress.     

Plant growth promotion by Bacillus megaterium involves cytokinin signaling

Accumulating evidence indicates that plant growth promoting rhizobacteria (PGPR) influence plant growth and development by the production of phytohormones such as auxins, gibberellins, and cytokinins. Little is known on the genetic basis and signal transduction components that mediate the beneficial effects of PGPRs in plants. We recently reported the identification of a Bacillus megaterium strain that promoted growth of A. thaliana and P. vulgaris seedlings. In this addendum, the role of cytokinin signaling in mediating the plant responses to bacterial inoculation was investigated using A. thaliana mutants lacking one, two or three of the putative cytokinin receptors CRE1, AHK2 and AHK3, and RPN12 a gene involved in cytokinin signaling. We show that plant growth promotion by B. megaterium is reduced in AHK2-2 single and double mutant combinations and in RPN12. Furthermore, the triple cytokinin-receptor CRE1-12/AHK2-2/AHK3-3 knockout was insensitive to inoculation in terms of growth promotion and root developmental responses. Our results indicate that cytokinin receptors play a complimentary role in plant growth promotion by B. megaterium.Key words: Arabidopsis, plant growth stimulation, root development, rhizobacteria     

The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg2?-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.     

Identification of amino acid substitutions that render the Arabidopsis cytokinin receptor histidine kinase AHK4 constitutively active

In Arabidopsis, three genes (AHK2, AHK3 and AHK4/CRE1) encode histidine kinases (His-kinases), which serve as cytokinin receptors. To understand how the external cytokinin signal activates the His-kinase across the cell membrane, we exploited the power of microbial genetics to isolate several AHK4 mutants that function independently of cytokinin in both prokaryotic and eukaryotic assay systems. In each mutant, a single amino acid substitution within the second membrane-spanning segment, or within the region around the phosphorylation His site, renders the His-kinase constitutively active. These mutant receptors appear to have a 'locked-on' conformation, even in the absence of stimulus. We discuss the implications of these data for the structure and function of the cytokinin receptor His-kinases in plants.     

Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism

We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.     

RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana

RanGAP is the GTPase-activating protein of the small GTPase Ran and is involved in nucleocytoplasmic transport in yeast and animals via the Ran cycle and in mitotic cell division. Arabidopsis thaliana has two copies of RanGAP, RanGAP1 and RanGAP2. To investigate the function of plant RanGAP, T-DNA insertional mutants were analysed. Arabidopsis plants with a null mutant of either RanGAP1 or RanGAP2 had no observable phenotype. Analysis of segregating progeny showed that double mutants in RanGAP1 and RanGAP2 are female gametophyte defective. Ovule clearing with differential interference contrast optics showed that mutant female gametophytes were arrested at interphase, predominantly after the first mitotic division following meiosis. In contrast, mutant pollen developed and functioned normally. These results show that the two RanGAPs are redundant and indispensable for female gametophyte development in Arabidopsis but dispensable for pollen development. Nuclear division arrest during a mitotic stage suggests a role for plant RanGAP in mitotic cell cycle progression during female gametophyte development.     

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase(NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Lossof-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion,our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.     

Analysis of the Maize dicer-like1 Mutant,fuzzy tassel,Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.     

Loss of cytosolic phosphoglucomutase compromises gametophyte development in Arabidopsis

Cytosolic phosphoglucomutase (cPGM) interconverts glucose-6-phosphate and glucose-1-phosphate and is a key enzyme of central metabolism. In this study, we show that Arabidopsis (Arabidopsis thaliana) has two cPGM genes (PGM2 and PGM3) encoding proteins with high sequence similarity and redundant functions. Whereas pgm2 and pgm3 single mutants were undistinguishable from the wild type, loss of both PGM2 and PGM3 severely impaired male and female gametophyte function. Double mutant pollen completed development but failed to germinate. Double mutant ovules also developed normally, but approximately half remained unfertilized 2 d after pollination. We attribute these phenotypes to an inability to effectively distribute carbohydrate from imported or stored substrates (e.g. sucrose) into the major biosynthetic (e.g. cell wall biosynthesis) and respiratory pathways (e.g. glycolysis and the oxidative pentose phosphate pathway). Disturbing these pathways is expected to have dramatic consequences for germinating pollen grains, which have high metabolic and biosynthetic activities. We propose that residual cPGM mRNA or protein derived from the diploid mother plant is sufficient to enable double mutant female gametophytes to attain maturity and for some to be fertilized. Mature plants possessing a single cPGM allele had a major reduction in cPGM activity. However, photosynthetic metabolism and growth were normal, suggesting that under standard laboratory conditions cPGM activity provided from one wild-type allele is sufficient to mediate the photosynthetic and respiratory fluxes in leaves.     

A Subset of Cytokinin Two-component Signaling System Plays a Role in Cold Temperature Stress Response in Arabidopsis

A multistep two-component signaling system is established as a key element of cytokinin signaling in Arabidopsis. Here, we provide evidence for a function of the two-component signaling system in cold stress response in Arabidopsis. Cold significantly induced the expression of a subset of A-type ARR genes and of GUS in Pro_ARR7:GUS transgenic Arabidopsis. AHK2 and AHK3 were found to be primarily involved in mediating cold to express A-type ARRs despite cytokinin deficiency. Cold neither significantly induced AHK2 and AHK3 expression nor altered the cytokinin contents of wild type within the 4 h during which the A-type ARR genes exhibited peak expression in response to cold, indicating that cold might induce ARR expression via the AHK2 and AHK3 proteins without alterations in cytokinin levels. The ahk2 ahk3 and ahk3 ahk4 mutants exhibited enhanced freezing tolerance compared with wild type. These ahk double mutants acclimated as efficiently to cold as did wild type. The overexpression of the cold-inducible ARR7 in Arabidopsis resulted in a hypersensitivity response to freezing temperatures under cold-acclimated conditions. The expression of C-repeat/dehydration-responsive element target genes was not affected by ARR7 overexpression as well as in ahk double mutants. By contrast, the arr7 mutants showed increased freezing tolerance. The ahk2 ahk3 and arr7 mutants showed hypersensitive response to abscisic acid (ABA) for germination, whereas ARR7 overexpression lines exhibited insensitive response to ABA. These results suggest that AHK2 and AHK3 and the cold-inducible A-type ARRs play a negative regulatory role in cold stress signaling via inhibition of ABA response, occurring independently of the cold acclimation pathway.     

Arabidopsis Histidine Kinase CKI1 Acts Upstream of HISTIDINE PHOSPHOTRANSFER PROTEINS to Regulate Female Gametophyte Development and Vegetative Growth

Cytokinin signaling is mediated by a multiple-step phosphorelay. Key components of the phosphorelay consist of the histidine kinase (HK)-type receptors, histidine phosphotransfer proteins (HP), and response regulators (RRs). Whereas overexpression of a nonreceptor-type HK gene CYTOKININ-INDEPENDENT1 (CKI1) activates cytokinin signaling by an unknown mechanism, mutations in CKI1 cause female gametophytic lethality. However, the function of CKI1 in cytokinin signaling remains unclear. Here, we characterize a mutant allele, cki1-8, that can be transmitted through female gametophytes with low frequency (∼0.17%). We have recovered viable homozygous cki1-8 mutant plants that grow larger than wild-type plants, show defective megagametogenesis and rarely set enlarged seeds. We found that CKI1 acts upstream of AHP (Arabidopsis HP) genes, independently of cytokinin receptor genes. Consistently, an ahp1,2-2,3,4,5 quintuple mutant, which contains an ahp2-2 null mutant allele, exhibits severe defects in megagametogenesis, with a transmission efficiency of <3.45% through female gametophytes. Rarely recovered ahp1,2-2,3,4,5 quintuple mutants are seedling lethal. Finally, the female gametophytic lethal phenotype of cki1-5 (a null mutant) can be partially rescued by IPT8 or ARR1 (a type-B Arabidopsis RR) driven by a CKI1 promoter. These results define a genetic pathway consisting of CKI1, AHPs, and type-B ARRs in the regulation of female gametophyte development and vegetative growth.     

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.     

SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.     

The peroxin loss-of-function mutation abstinence by mutual consent disrupts male-female gametophyte recognition

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.     

Phenotypic Characterization of a Female Sterile Mutant in Rice

A female sterile mutant, derived from a spontaneous mutation, was first discovered In rlce （Oryzs satlvs L. sep. Indlca） restorer llne 202R. Wlth normal fiowerlng, the mutant exhlblts an extremely low seed-sattlng rate. When the mutant Is crossed as a pollen donor, the seeds set normally; whereas when It Is used as a pollen recelver, no seeds are obtalned even wlth mlxed pollen gralns of dlfferent varletles sprlnkled over the atlgmas. The fioret of the mutant, conslatlng of slx stamens and one platll, looks the same as that of the wlld type In the malefemale organs, except that less than 10% of the mutant florets have three atlgmas on the ovary. Although the mutant has a low seed-setting rate, Its pollen fertility Is approximately 87.1%, which Is equal to that of the wild type. In addition, more than 90% of the mature embryo sacs of the mutant have complete Inner structures. At every stage after pollination, the sperm, embryo, and endosperm are not found In the mutant embryo sac, whereas the disintegration of the egg cell that does not accomplish fertilization Is visible. Through observetlons with a fluorescence microscope, we have found that the pollen grains germinate normally, whereas the pollen tube abnormally elongates in the style-transmitting tissue. The mutant pollen tubes display various defects In the style, such as slower elongation, conversed elongation, distorted elongation, swollen tips, or branched tips. As a result, the growth of the pollen tubes ceases In the style, and, therefore, the pollen tubes cannot reach the embryo sac and the process of double fertilization Is blocked. Based on these observations, we conclude that this mutant, designated as fs-202R, Is a novel type of female sterile mutation In rice, which causes the arrest of the elongation of the pollen tube.