Evolutionarily diverse SYP1 Qa‐SNAREs jointly sustain pollen tube growth in Arabidopsis

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans‐SNARE complexes. Qa‐SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen‐specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen‐specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa‐SNARE proteins to sustain their growth‐promoting membrane dynamics during the reproductive process.     

Long‐chain base phosphates modulate pollen tube growth via channel‐mediated influx of calcium

Long‐chain base phosphates (LCBPs) have been correlated with amounts of crucial biological processes ranging from cell proliferation to apoptosis in animals. However, their functions in plants remain largely unknown. Here, we report that LCBPs, sphingosine‐1‐phosphate (S1P) and phytosphingosine‐1‐phosphate (Phyto‐S1P), modulate pollen tube growth in a concentration‐dependent bi‐phasic manner. The pollen tube growth in the stylar transmitting tissue was promoted by SPHK1 overexpression (SPHK1‐OE) but dampened by SPHK1 knockdown (SPHK1‐KD) compared with wild‐type of Arabidopsis; however, there was no detectable effect on in vitro pollen tube growth caused by misexpression of SPHK1. Interestingly, exogenous S1P or Phyto‐S1P applications could increase the pollen tube growth rate in SPHK1‐OE, SPHK1‐KD and wild‐type of Arabidopsis. Calcium ion (Ca²⁺)‐imaging analysis showed that S1P triggered a remarkable increase in cytosolic Ca²⁺ concentration in pollen. Extracellular S1P induced hyperpolarization‐activated Ca²⁺ currents in the pollen plasma membrane, and the Ca²⁺ current activation was mediated by heterotrimeric G proteins. Moreover, the S1P‐induced increase of cytosolic free Ca²⁺ inhibited the influx of potassium ions in pollen tubes. Our findings suggest that LCBPs functions in a signaling cascade that facilitates Ca²⁺ influx and modulates pollen tube growth.     

Four Arabidopsis berberine bridge enzyme‐like proteins are specific oxidases that inactivate the elicitor‐active oligogalacturonides

Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H₂O₂‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.     

Functional analysis of related CrRLK1L receptor‐like kinases in pollen tube reception

The Catharanthus roseus Receptor‐Like Kinase 1‐like (CrRLK1L) family of 17 receptor‐like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin‐like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co‐factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception.     

Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development,pollen tube guidance and embryogenesis

Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2β revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock‐out mutants of atipk2α and atipk2β were indistinguishable from the wild type, whereas the atipk2α atipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2α atipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity‐altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS‐ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS‐ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi‐in vitro, whereas normal PT growth was shown in its sense control in vitro/semi‐in vitro. PT growth was impaired in a manner dependent on the dose of AS‐ODNs against both ANX1 and ANX2 above 10 μm . The treatment with AS‐ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS‐ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS‐ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS‐ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.     

Patterns and sources of variation in pollen deposition and pollen tube formation in flowers of the endemic monoecious shrub Cnidoscolus souzae (Euphorbiaceae)

Pollen deposition and pollen tube formation are key components of angiosperm reproduction but intraspecific variation in these has rarely been quantified. Documenting and partitioning (populations, plants and flowers) natural variation in these two aspects of plant reproduction can help uncover spatial mosaics of reproductive success and underlying causes. In this study, we assess variation in pollen deposition and pollen tube formation for the endemic monoecious shrub Cnidoscolus souzae throughout its distribution range in Mexico, and determine how this variation is structured among populations, plants and flowers. We also infer the relative importance of pollen quantity and quality in determining pollination success in this species. While we found no evidence suggesting that pollen receipt limits C. souzae reproduction across 19 populations, we did find extensive variation in pollen load size and pollen tube number per flower. Total variation in pollen receipt and pollen tube number was mostly explained by intra‐individual and among‐population variance. Furthermore, pollen load size had a stronger effect on the number of pollen tubes at the base of the style than pollen germination rate, suggesting that pollen quantity may be more important than quality for pollen tube success in C. souzae. Our results suggest that both small within‐plant flower differences and broad‐scale differences in community attributes can play an important role in determining pollination success. We emphasise the need to evaluate patterns and sources of variation in pollen deposition and pollen tube formation as a first step in understanding the causes of variation in pollination success over broad spatial scales.     

An oligogalacturonide‐derived molecular probe demonstrates the dynamics of calcium‐mediated pectin complexation in cell walls of tip‐growing structures

Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell‐to‐cell adhesion. Current immunological methods enable only steady‐state detection of egg box formation in situ. Here we present a tool for efficient real‐time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium‐mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de‐esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real‐time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip‐growing structures.     

Cadmium‐induced and trans‐generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana

Trans‐generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well‐known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd‐exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd‐exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.     

The glucosinolate breakdown product indole‐3‐carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana

The glucosinolate breakdown product indole‐3‐carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole‐3‐carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole‐3‐carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole‐3‐carbinol rapidly and reversibly inhibits root elongation in a dose‐dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole‐3‐carbinol and the auxin perception machinery was suggested, as application of indole‐3‐carbinol rescues auxin‐induced root phenotypes. In vitro and yeast‐based protein interaction studies showed that indole‐3‐carbinol perturbs the auxin‐dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3‐indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole‐3‐carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole‐3‐carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

Homology‐dependent repair is involved in 45S rDNA loss in plant CAF‐1 mutants

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology‐dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock‐out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B‐dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double‐stranded breaks (measured as γ‐H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S‐phase, and is ATM‐independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non‐transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double‐stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single‐stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.     

Structural,mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water‐mediated mechanism

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2‐fucosyl residues to xyloglucan side chains – a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X‐ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X‐ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water‐mediated fucosylation mechanism facilitated by an H‐bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.