Teh-hui Kao

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.

Identification of the pollen determinant of S-RNase-mediated self-incompatibility

Many flowering plants have adopted self-incompatibility mechanisms to prevent inbreeding and promote out-crosses. In the Solanaceae, Rosaceae and Scrophulariaceae, two separate genes at the highly polymorphic S-locus control self-incompatibility interactions: the S-RNase gene encodes the pistil determinant and the previously unidentified S-gene encodes the pollen determinant. S-RNases interact with pollen S-allele products to inhibit the growth of self-pollen tubes in the style. Pollen-expressed F-box genes showing allelic sequence polymorphism have recently been identified near to the S-RNase gene in members of the Rosaceae and Scrophulariaceae; but until now have not been directly shown to encode the pollen determinant. Here we report the identification and characterization of PiSLF, an S-locus F-box gene of Petunia inflata (Solanaceae). We show that transformation of S1S1, S1S2 and S2S3 plants with the S2-allele of PiSLF causes breakdown of their pollen function in self-incompatibility. This breakdown of pollen function is consistent with ‘competitive interaction’, in which pollen carrying two different pollen S-alleles fails to function in self-incompatibility. We conclude that PiSLF encodes the pollen self-incompatibility determinant.

Changes in root cap pH are required for the gravity response of the Arabidopsis root

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide–Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.

The Molecular and Genetic Bases of S-RNase-Based Self-Incompatibility

The majority of flowering plants produce perfect flowers that contain both the male and female reproductive organs in close proximity; consequently, they would have a strong tendency to self-fertilize if there were no mechanisms to prevent them from doing so. Because inbreeding can result in reduced

Petunia Phospholipase C1 Is Involved in Pollen Tube Growth

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)–cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC δ1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.

Collaborative non-self recognition system in S-RNase-based self-incompatibility.

Dissecting Self-Incompatibility Although the pollen may be available for a flower to fertilize itself, molecular determinants on the pollen and the pistil prevent inbreeding in a process termed self-incompatibility. In the Petunia self-incompatibility, if male determinants (F-box proteins) on pollen are recognized by a female ribonuclease determinant on the pistil, the pollen tube is killed when its ribosomal RNA is digested. Outcrossed fertilizations can occur because of allelic diversity in the female that fails to recognize its male counterparts; however, the genetic diversity of the ribonuclease gene is greater than that of the known F-box gene. Kubo et al. (p. 796; see the Perspective by Indriolo and Goring) have discovered that there are several related F-box genes in Petunia, each of which brings its own allelic diversity to bear—thus, increasing the variety of potential mating partners. A highly diverse set of (male) pollen-determinant proteins in Petunia recognizes non-self (female) pistil determinants. Self-incompatibility in flowering plants prevents inbreeding and promotes outcrossing to generate genetic diversity. In Solanaceae, a multiallelic gene, S-locus F-box (SLF), was previously shown to encode the pollen determinant in self-incompatibility. It was postulated that an SLF allelic product specifically detoxifies its non-self S-ribonucleases (S-RNases), allelic products of the pistil determinant, inside pollen tubes via the ubiquitin–26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia, at least three types of divergent SLF proteins function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase.

From a pollen tube cDNA library of Petunia inflata, we isolated clones encoding a protein with structural features and biochemical properties characteristic of receptor-like kinases. It was designated PRK1 for pollen receptor-like kinase 1. The cytoplasmic domain of PRK1 is highly similar to the kinase domains of other plant receptor-like kinases and contains nearly all of the conserved amino acids for serine/threonine kinases. The extracellular domain of PRK1 contains leucine-rich repeats as found in some other plant receptor-like kinases, but overall its sequence in this region does not share significant similarity. Characterization of a gene encoding PRK1 revealed the presence of two introns. During pollen development, PRK1 mRNA was first detected in anthers containing mostly binucleate microspores; it reached the highest level of mature pollen and remained at a high level in in vitro-germinated pollen tubes. The recombinant cytoplasmic domain of PRK1 autophosphorylated on serine and tyrosine, suggesting that PRK1 may be a dual-specificity kinase. Monospecific immune serum to the recombinant extracellular domain of PRK1 detected a 69-kD protein in microsomal membranes of pollen and pollen tubes. The characteristics of PRK1 suggest that it may play a role in signal transduction events during pollen development and/or pollination.

Primary structural features of the self-incompatibility protein in solanaceae

SummaryWe present a sequence comparison of 12 S-allele-associated proteins from three solanaceous species with gametophytic self-incompatibility: Nicotiana alata, Petunia inflata, and Solanum chacoense. The allelic variants of the S-protein exhibit a very high degree of sequence diversity consistent with their function as recognition molecules. We identify 41 perfectly conserved residues, 18 of which are also conserved in two fungal ribonucleases, RNase T2 and RNase Rh. The residues conserved in both the S-proteins and the ribonucleases include two histidines essential for catalysis, four cysteines involved in disulfide bridges, and hydrophobic residues probably involved in the core structure of the proteins. This conservation between the two ribonucleases and the 12 divergent S-proteins confirms the previously recognized similarity between 3 more closely related N. alata S-proteins and these ribonucleases, and argues strongly for the functional importance of the ribonuclease activity of the S-protein in self-incompatibility. We also identify the 19 most variable residues, which are the prime candidates for the S-allele-specificity determinant. Twelve of these nineteen residues are clustered in two regions of hypervariability, designated HVa and HVb, which are also the most prominent hydrophilic regions of the S-protein. We suggest that these two regions might form parts of the putative pollen recognition site. Identification of these structural features forms a foundation for the study of the molecular basis of self-recognition and the biochemical mechanism of inhibition of self-pollen tube growth.

Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S1-, S2-, and S3-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S3-RNase is not degraded to any significant extent; however, deglycosylated S3-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

SummaryWe have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.