Danilo D. Fernando

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.

Transient gene expression in pine pollen tubes following particle bombardment

Abstract A biolistic particle delivery system was used to genetically transform pollen tubes of three species of white pine (Pinus aristata, P. griffithii and P. monticola). The introduced plasmid DNA contained the GUS coding sequence flanked by the 35S CaMV promoter and NOS terminator sequences. Successful gene delivery was demonstrated by transient GUS expression as evaluated by standard histochemical assay. Distance of target specimens significantly influenced transient GUS expression in all three species of white pine. A target distance of 6 cm resulted in a significant number of transformed pollen tubes in P. aristata and P. griffithii, while distances of 6 and 9 cm resulted in a significant number of transformed pollen tubes in P. monticola. Generally, the number of pollen tubes expressing GUS activity was higher in P. aristata than in P. griffithii and P. monticola. The possibility of using GUS-transformed pollen tubes in conjunction with in vitro fertilization in conifers was examined. Gene expression in pollen tubes was also examined under electron microscopy where the X-glu reaction product occurred as large crystalline electron-dense precipitates in the cytoplasm.

IDENTIFICATION OF A CHLOROPLAST DEHYDRIN IN LEAVES OF MATURE PLANTS

Several types of proteins are known to accumulate as a result of dehydration stress in plants, and many of these are thought to serve a protective function. This includes the dehydrin family of proteins, which accumulate in cells in response to drought, low temperatures, or salinity and in embryo tissues during the maturation phase of seed development, when the seed is losing water in preparation for dormancy. Many studies to date have concentrated on the expression, localization, and function of dehydrins in seed tissues. Our study provides some of the first evidence for a chloroplast‐localized dehydrin by using cell fractionation combined with immunofluorescence and immunogold electron microscopy to determine dehydrin location in mature leaf tissues of Pisum sativum and Zea mays. This article also documents constitutive expression of the chloroplast dehydrin as well as expression during different dehydrative stresses. The chloroplast‐dehydrin expression pattern differs from most other dehydrins studied to date and suggests a role in basic cell metabolism for this particular dehydrin.

Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T1 lines were obtained, and a 3:1 segregation ratio was obtained in the T2 generation of each line. In the T3 generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.

In vitro pollen tube growth and penetration of female gametophyte in Douglas fir (Pseudotsuga menziesii)

Abstract Pollen tube and female gametophyte interactions in Douglas fir (Pseudotsuga menziesii) were examined in vitro. Formation of pollen tubes in Douglas fir occurred on a modified Murashige and Skoog medium in which concentrations of H3BO3 and Ca(NO3)2 were altered and supplemented with sucrose and polyethylene glycol. Addition of 100 μg/ml H3BO3 and 300 μg/ml Ca(NO3)2 resulted in optimum pollen viability. Lack of H3BO3 inhibited pollen tube formation. Addition of H3BO3 and Ca(NO3)2 significantly increased pollen tube formation within one week in culture. Using a medium supplemented with mannitol, viability of Douglas fir pollen can be sustained for 7 weeks in culture, about the same length of time as in vivo. However, pollen tubes are not formed. This suggests that the factors responsible for tube formation reside in the external environment of the pollen. Culture of female gametophytes to examine egg viability and longevity had not been done previously. We found that egg viability in culture is short-lived, and therefore the window to study and manipulate events of fertilization in Douglas fir is very limited. In spite of this, about 7% of the female gametophytes that were co-cultured became penetrated by pollen tubes. In vitro archegonial penetration has been repeatedly achieved, but pollen tubes also penetrated other parts of the female gametophytes. Pollen tubes also penetrated non-viable eggs. Most female gametophytes were not penetrated because of pollen tube branching and swelling, failure of tubes to orient towards the female gametophytes, or premature pollen tube death due to plasmolysis. This report outlines the first attempt towards in vitro fertilization in conifers.

Anatomical aspects of angiosperm root evolution.

BACKGROUND AND AIMS Anatomy had been one of the foundations in our understanding of plant evolutionary trends and, although recent evo-devo concepts are mostly based on molecular genetics, classical structural information remains useful as ever. Of the various plant organs, the roots have been the least studied, primarily because of the difficulty in obtaining materials, particularly from large woody species. Therefore, this review aims to provide an overview of the information that has accumulated on the anatomy of angiosperm roots and to present possible evolutionary trends between representatives of the major angiosperm clades. SCOPE This review covers an overview of the various aspects of the evolutionary origin of the root. The results and discussion focus on angiosperm root anatomy and evolution covering representatives from basal angiosperms, magnoliids, monocots and eudicots. We use information from the literature as well as new data from our own research. KEY FINDINGS The organization of the root apical meristem (RAM) of Nymphaeales allows for the ground meristem and protoderm to be derived from the same group of initials, similar to those of the monocots, whereas in Amborellales, magnoliids and eudicots, it is their protoderm and lateral rootcap which are derived from the same group of initials. Most members of Nymphaeales are similar to monocots in having ephemeral primary roots and so adventitious roots predominate, whereas Amborellales, Austrobaileyales, magnoliids and eudicots are generally characterized by having primary roots that give rise to a taproot system. Nymphaeales and monocots often have polyarch (heptarch or more) steles, whereas the rest of the basal angiosperms, magnoliids and eudicots usually have diarch to hexarch steles. CONCLUSIONS Angiosperms exhibit highly varied structural patterns in RAM organization; cortex, epidermis and rootcap origins; and stele patterns. Generally, however, Amborellales, magnoliids and, possibly, Austrobaileyales are more similar to eudicots, and the Nymphaeales are strongly structurally associated with the monocots, especially the Acorales.

RNA and protein synthesis during in vitro pollen germination and tube elongation in Pinusmonticola and other conifers

Abstract Pollen germination and tube elongation in Pinusmonticola are accompanied by RNA and protein synthesis as shown by the effects of inhibitors such as actinomycin D and cycloheximide, respectively. Pollen grains germinate in the presence of actinomycin D, but further tube elongation is inhibited. This suggests that RNAs needed for germination are already available in the mature ungerminated pollen, but continued tube elongation depends on the synthesis of new RNAs. Using cycloheximide, our results indicate that some proteins essential for germination and tube elongation are not yet available in the mature ungerminated pollen. In P. monticola, it appears that these proteins are synthesized at the onset of pollen germination and during tube elongation. The effects of inhibiting transcription and translation in eight other conifers are the same as in P. monticola, suggesting a common trend. In P. monti-cola, profiles of pollen grains and pollen tubes varied in the expression of at least ten proteins. Based on the stages examined, the protein profiles of 2-day-old tubes appear to be the most variable with some proteins increasing or decreasing in intensity only at this stage. In P. monticola, four proteins (26, 27, 38 and 40 kDa) are differentially expressed during pollen tube development. The most notable is a 26-kDa protein which is specifically expressed in pollen tubes. It is possible that this protein controls a function unique to pollen tubes. This report adds to our knowledge of the regulation of pollen tubes development in conifers. It also offers insights on why development of pollen tubes in conifers takes much longer than in flowering plants.

Sexual reproduction and crossing barriers in white pines: the case between Pinus lambertiana (sugar pine) and P. monticola (western white pine)

The sexual reproductive process in Pinus lambertiana has not been completely described, and previous attempts to generate hybrids with Pinus monticola and other North American pines have not been successful. The nature of incompatibility barriers between P. lambertiana and P. monticola is unknown. This needs to be understood if strategies are to be developed to overcome the said barriers. In this paper, development on interspecific crosses is compared with that on intraspecific crosses on the same parent trees. Pollen grains of both species germinated on the nucellus of both species within a week after pollination. Seed cone receptivity in P. lambertiana came approximately 2 weeks after receptivity in P. monticola, and this delay was perpetuated throughout ovule development in the first year of the reproductive process. Development of the second-year seed cones proceeded more gradually in P. lambertiana. However, seed cones reached maturity only for P. monticola x P. lambertiana. In both crosses, the barriers to hybridization occurred during the second year of the reproductive process. With the P. lambertiana as the seed parent, it was manifested through the failure of the megaspores at the free-nuclear stage to resume development. When P. monticola was used as the seed parent, the male and female gametes failed to fuse. Our results clearly show that the barriers to hybridization in these species occur before or at fertilization. However, the exact mechanisms behind these are still unknown. Based on the results of this study, we present several strategies to bypass the developmental barriers and possibly produce hybrid progenies.

Development and Structure of Ovule, Embryo Sac, Embryo, and Endosperm in Butomus umbellatus (Butomaceae)

Studies of reproductive structures are important in understanding developmental processes and taxonomic and phylogenetic relationships of species In spite of the data available in the literature on Butomus umbellatus, many important characters have not been extensively studied and properly used in the evaluation of its relationships. This study has clarified several conflicting reports on the female reproductive structures in B. umbellatus, including (1) ability of the archesporial cells to produce parietal cells, (2) crassinucellate type of ovule, (3) timing of ovule curvature, (4) caryophyllad type of embryogenesis different from the Sagittaria variation, (5) cellularized antipodals, (6) presence of wall ingrowths in the synergids, and (7) nature of the horizontal partition of the helobial endosperm. Many of these features provide further support to the acceptance of the unspecialized condition and monogeneric status of the Butomaceae. Therefore, B. umbellatus may be the least specialized among the Alismatales. Although this study provides clearer factual bases for morphological, systematic, and phylogenetic inferences, a thorough reexamination of reproductive structures in other members of the Alismatales should be done before any attempt on phylogenetic analysis can be made.

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.