Gibberellins are required for embryo growth and seed development in pea

The gibberellin (GA) biosynthesis mutants lh-1 and lh-2 have been used to examine the physiological role of GAs in pea seed development. The LH protein is required for the three-step oxidation of ent -kaurene to ent -kaurenoic acid early in the GA biosynthesis pathway. The allele-specific interaction of lh-1 and lh-2 with chemical inhibitors of these three steps suggests that LH encodes the multi-functional GA biosynthesis enzyme ent -kaurene oxidase. Unlike the lh-2 mutation which reduces seed weight and decreases seed survival by ∼50% compared with wild-type plants, the lh-1 allele has a transient effect on embryo and seed growth and only slightly increases seed abortion. These seed phenotypes parallel the effects of the two mutant alleles on GA levels in young seeds. Detailed examination of the growth of lh-1 seeds reveals homeostatic regulation of GA-promoted embryo and seed growth. Although GA-deficient seeds grow more slowly than WT seeds, decreased assimilate availability to the developing seeds is not the primary reason for the altered seed development. Instead, GAs act to promote some process(es) required for embryo and seed growth and only indirectly influence the distribution of assimilates. How GA deficiency causes seed abortion is not known but it may simply be a consequence of reduced seed or embryo growth rate. These results demonstrate that even relatively small changes in the levels of GAs in young seeds can alter seed development and suggest that the available GA-related mutants may represent only a subset of all possible mutants with reduced GA levels or GA signalling.     

Cdi gene is required for pollen germination and tube growth in Arabidopsis

Pollen germination and tube growth are of essential importance to sexual reproduction of flowering plants. Several biological processes including cell wall biosynthesis and modification are known to be involved in pollen tube growth, though the underlying molecular mechanisms remain largely to be investigated. Here we report the identification and functional characterization of the Arabidopsis gene Cdi, which encodes a putative nucleotide-diphospho-sugar transferase. Cdi is preferentially expressed in pollen grains and pollen tubes. Transient expression of Cdi:GFP fusion protein showed that CDI is localized in the cytosol. Mutation in Cdi impaired pollen germination and pollen tube growth thus leading to disrupted male transmission. These results suggest that Cdi is an essential gene required for pollen germination and tube growth.     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

Gibberellins are not required for normal stem growth in Arabidopsis thaliana in the absence of GAI and RGA

The growth of Arabidopsis thaliana is quantitatively regulated by the phytohormone gibberellin (GA) via two closely related nuclear GA-signaling components, GAI and RGA. Here we test the hypothesis that GAI and RGA function as "GA-derepressible repressors" of plant growth. One prediction of this hypothesis is that plants lacking GAI and RGA do not require GA for normal stem growth. Analysis of GA-deficient mutants lacking GAI and RGA confirms this prediction and suggests that in the absence of GAI and RGA, "growth" rather than "no growth" is the default state of plant stems. The function of the GA-signaling system is thus to act as a control system regulating the amount of this growth. We also demonstrate that the GA dose dependency of hypocotyl elongation is altered in mutants lacking GAI and RGA and propose that increments in GAI/RGA repressor function can explain the quantitative nature of GA responses.     

Metabolism and roles of phosphatidylinositol 3-phosphate in pollen development and pollen tube growth in Arabidopsis

Phosphoinositides play important roles in eukaryotic cells, although they constitute a minor fraction of total cellular lipids. Specific kinases and phosphatases function on the regulation of phosphoinositide levels. Phosphatidylinositol 3-phosphate (PtdIns3P), a molecule of phosphoinositides regulates multiple aspects of plant growth and development. In this article, we introduce and discuss the kinases and phosphatases involved in PtdIns3P metabolism and their roles in pollen development and pollen tube growth in Arabidopsis.     

Actin bundler PLIM2s are involved in the regulation of pollen development and tube growth in Arabidopsis

Microspores develop inside the anther, where they are surrounded by nourishing tapetal cells. However, many cellular processes occurring during microspore development in the locule are poorly characterized. The actin cytoskeleton is known to play a crucial role in various aspects of the plant developmental process. During pollen tube tip growth, actin cytoskeleton serves as an efficient molecular transportation track, although how it functions in pollen development is unknown. The plant actin bundler PLIM2s have been shown to regulate actin bundling in different cells. Here, we investigate the biological function of three Arabidopsis pollen-specific LIM proteins, PLIM2a, PLIM2b, and PLIM2c (collectively, PLIM2s), in pollen development and tube growth. Variable degrees of suppressed expression of the PLIM2s by RNA interference resulted in aberrant phenotypes. Complete suppression of the PLIM2s totally disrupted pollen development, producing abortive pollen grains and rendering the transgenic plants sterile. Partial suppression of the PLIM2s arrested pollen tube growth to a lesser extent, resulting in short and swollen pollen tubes. Finally, the PLIM2c promoter initiated expression in pollen during stamen filament elongation, and the PLIM2c protein was located on particle structures in the developing pollen grains in Arabidopsis. These suggest that the actin bundler, PLIM2s, are an important factor for Arabidopsis pollen development and tube growth.     

Cytological study of pollen tube growth and early seed development inPetunia inflata

Pollen tube growth from the stigma into the ovule, and the early fruit and seed development following fertilization were examined using fluorescence microscopy, scanning electron microscopy and light microscopy inPetunia inflata. After growing intercellularly in the transmitting tract for 24–36 hr, the pollen tubes emerged into the top part of the ovary cavity and grew along the surface of the septum to reach the ovule. It grew around the furnicle and penetrated the micropyle to enter the embryo sac for fertilization. After fertilization, the endosperm nucleus divided first before the embryo, and the cell wall formation occurred following the division, exhibiting the pattern of cellular type of endosperm development. The first division of the zygote did not occur until 3 days after pollination. At 6 days after pollination, the seeds grew considerably and the endosperm has gone through multiple rounds of cell division. High starch formation in the integument, especially around the embryo sac, was also observed.     

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1‐3 pskr2‐1 and tpst‐1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1‐3 pskr2‐1 and of tpst‐1 were shorter. In planta, growth of wild‐type pollen in pskr1‐3 pskr2‐1 and tpst‐1 flowers appeared slower than growth in wild‐type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1‐3 pskr2‐1 and in tpst‐1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross‐pollination experiments with wild‐type, pskr1‐3 pskr2‐1 and tpst‐1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.     

Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth

While mammals and fungi possess nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) isoforms, Arabidopsis thaliana only contains a single NMNAT gene, AtNMNAT (At5g55810). We analyzed the enzymatic activity of the AtNMNAT-encoded protein to determine the role of AtNMNAT in plant development. AtNMNAT catalyzed the synthesis of nicotinate adenine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Preiss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. Moreover, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decrease in NAD content in pollen. Cytological examinations revealed that the atnmnat mutant was gametophytically impaired in in vivo and in vitro pollen tube growth. Our results suggest that metabolic fulfillment via the NAD pathway is indispensable for normal pollen growth and subsequent normal seed production.     

Molecular evidence that most RNAs required for germination and pollen tube growth are stored in the mature pollen grain in petunia

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.     

A lectin receptor-like kinase is required for pollen development in Arabidopsis

Lectin receptor-like kinases (Lectin RLKs) are a large family of receptor-like kinases with an extracellular legume lectin-like domain. There are approximately 45 such receptor kinases in Arabidopsis thaliana. Surprisingly, although receptor-like kinases in general are well investigated in Arabidopsis, relatively little is known about the functions of members of the Lectin RLK family. A number of studies implicated members of this family in various functions, such as disease resistance, stress responses, hormone signaling, and legume-rhizobium symbiosis. Our current work demonstrated that mutation in one Lectin RLK gene led to male sterility in Arabidopsis. The sterility was due to defects in pollen development. Pollen development proceeded normally in the mutant until anther stage 8. After that, all pollen grains deformed and collapsed. Mature pollen grains were much smaller than wild-type pollen grains, glued together, and totally collapsed. Therefore, the mutant was named sgc, standing for small, glued-together, and collapsed pollen mutant. The mutant phenotype appeared to be caused by an unidentified sporophytic defect due to the mutation. As revealed by analysis of the promoter-GUS transgenic plants and the gene expression analysis using RT-PCR, the gene showed an interesting temporal and spatial expression pattern: it had no or a low expression in young flowers (roughly before anther stage 6), reached a maximum level around stages 6-7, and then declined gradually to a very low level in young siliques. No expression was detected in microspores or pollen. Together, our data demonstrated that SGC Lectin RLK plays a critical role in pollen development.     

Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis

Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall-associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.     

Serine esterases are required for pollen tube penetration of the stigma in Brassica

. We have investigated the diversity of serine esterases in pollen and stigma tissues of Brassica napus and the role of these enzymes in pollen germination and pollen tube penetration of the stigma. The serine esterase-specific inhibitor diisopropyl fluorophosphate was used as a probe in a tritiated form, [³H]-DIPF, to determine the number and diversity of serine esterases in crude protein extracts from pollen and stigma. Seven serine esterases were identified in pollen and at least seven serine esterases were identified in stigma. The most abundant enzymes had molecular weights of 30-50 kDa. In the pollen extract a serine esterase was detected with the same molecular weight, 22 kDa, as an esterase previously shown to be a cutinase. Only one serine esterase (40 kDa) appeared to be shared between pollen and stigma extracts. Butyrate esterase activity in pollen and stigma extracts was assayed using p-nitrophenyl butyrate (PNB), an ester substrate frequently used in 'cutinase' assays. Total PNBase activity in pollen and stigma extracts was shown to be significantly reduced by the serine esterase inhibitors DIPF and ebelactone B. When DIPF and ebelactone B were applied to stigmas prior to pollination, pollen germination was not significantly affected but, at the highest inhibitor concentrations, up to 70% of germinating pollen tubes failed to penetrate the stigma surface. These data demonstrate that serine esterases, most probably cutinase(s), are required for pollen tube penetration of the dry cuticularised Brassica stigma.     

Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis

The metal chelator nicotianamine promotes the bioavailability of Fe and reduces cellular Fe toxicity. For breeding Fe-efficient crops, we need to explore the fundamental impact of nicotianamine on plant development and physiology. The quadruple nas4x-2 mutant of Arabidopsis thaliana cannot synthesize any nicotianamine, shows strong leaf chlorosis, and is sterile. To date, these phenotypes have not been fully explained. Here, we show that sink organs of this mutant were Fe deficient, while aged leaves were Fe sufficient. Upper organs were also Zn deficient. We demonstrate that transport of Fe to aged leaves relied on citrate, which partially complemented the loss of nicotianamine. In the absence of nicotianamine, Fe accumulated in the phloem. Our results show that rather than enabling the long-distance movement of Fe in the phloem (as is the case for Zn), nicotianamine facilitates the transport of Fe from the phloem to sink organs. We delimit nicotianamine function in plant reproductive biology and demonstrate that nicotianamine acts in pollen development in anthers and pollen tube passage in the carpels. Since Fe and Zn both enhance pollen germination, a lack of either metal may contribute to the reproductive defect. Our study sheds light on the physiological functions of nicotianamine.