Genome-Wide Analysis of the BAHD Family in Welsh Onion and CER2-LIKEs Involved in Wax Metabolism.

BAHD acyltransferases (BAHDs), especially those present in plant epidermal wax metabolism, are crucial for environmental adaptation. Epidermal waxes primarily comprise very-long-chain fatty acids (VLCFAs) and their derivatives, serving as significant components of aboveground plant organs. These waxes play an essential role in resisting biotic and abiotic stresses. In this study, we identified the BAHD family in Welsh onion (Allium fistulosum). Our analysis revealed the presence of AfBAHDs in all chromosomes, with a distinct concentration in Chr3. Furthermore, the cis-acting elements of AfBAHDs were associated with abiotic/biotic stress, hormones, and light. The motif of Welsh onion BAHDs indicated the presence of a specific BAHDs motif. We also established the phylogenetic relationships of AfBAHDs, identifying three homologous genes of CER2. Subsequently, we characterized the expression of AfCER2-LIKEs in a Welsh onion mutant deficient in wax and found that AfCER2-LIKE1 plays a critical role in leaf wax metabolism, while all AfCER2-LIKEs respond to abiotic stress. Our findings provide new insights into the BAHD family and lay a foundation for future studies on the regulation of wax metabolism in Welsh onion.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Cell type-specific gene expression underpins remodelling of cell wall pectin in exocarp and cortex during apple fruit development.

In apple (Malus×domestica) fruit, the different layers of the exocarp (cuticle, epidermis, and hypodermis) protect and maintain fruit integrity, and resist the turgor-driven expansion of the underlying thin-walled cortical cells during growth. Using in situ immunolocalization and size exclusion epitope detection chromatography, distinct cell type differences in cell wall composition in the exocarp were revealed during apple fruit development. Epidermal cell walls lacked pectic (1→4)-ß-d-galactan (associated with rigidity), whereas linear (1→5)-α-l-arabinan (associated with flexibility) was exclusively present in the epidermal cell walls in expanding fruit and then appeared in all cell types during ripening. Branched (1→5)-α-l-arabinan was uniformly distributed between cell types. Laser capture microdissection and RNA sequencing (RNA-seq) were used to explore transcriptomic differences controlling cell type-specific wall modification. The RNA-seq data indicate that the control of cell wall composition is achieved through cell-specific gene expression of hydrolases. In epidermal cells, this results in the degradation of galactan side chains by possibly five ß-galactosidases (BGAL2, BGAL7, BGAL10, BGAL11, and BGAL103) and debranching of arabinans by α-arabinofuranosidases AF1 and AF2. Together, these results demonstrate that flexibility and rigidity of the different cell layers in apple fruit during development and ripening are determined, at least in part, by the control of cell wall pectin remodelling.

Active rearrangements in the cell wall follow polymer concentration during postharvest withering in the berry skin of Vitis vinifera cv. Corvina.

During grape postharvest withering, a worldwide practice used to produce important high-quality wines, the solute concentration increases due to dehydration, and many organoleptic and quality traits, especially related to the berry skin, are affected in a cultivar-specific manner. Nevertheless, a complete comprehension of the underlying processes is still lacking. In this work, we applied ATR-FTIR micro-spectroscopy combined with PCA to monitor cell wall biochemical changes at three stages during postharvest withering on the internal and external sides of the berry skin of the Vitis vinifera cv. Corvina, an important local variety of the Verona province in Italy. The obtained results were integrated by profiling xylogucans and pectins through immunohistochemistry and by genome-wide transcriptomic analysis performed at the same withering stages. Our analysis indicates a gradual passive polymer concentration due to water loss in the first two months of postharvest withering, followed by active structural modifications in the last month of the process. Such rearrangements involve xyloglucans in the internal surface, cuticle components and cellulose in the external surface, and pectins in both surfaces. Moreover, by investigating the expression trend of cell wall metabolism-related genes, we identified several putative molecular markers associated to the polymer dynamics. The present study represents an important step towards an exhaustive comprehension of the postharvest withering process, which is of great interest from both the biological and technological points of view.

The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua.

The glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood. To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants. Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis. Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant.

Potato native and wound periderms are differently affected by down-regulation of FHT, a suberin feruloyl transferase.

Potato native and wound healing periderms contain an external multilayered phellem tissue (potato skin) consisting of dead cells whose cell walls are impregnated with suberin polymers. The phellem provides physical and chemical barriers to tuber dehydration, heat transfer, and pathogenic infection. Previous RNAi-mediated gene silencing studies in native periderm have demonstrated a role for a feruloyl transferase (FHT) in suberin biosynthesis and revealed how its down-regulation affects both chemical composition and physiology. To complement these prior analyses and to investigate the impact of FHT deficiency in wound periderms, a bottom-up methodology has been used to analyze soluble tissue extracts and solid polymers concurrently. Multivariate statistical analysis of LC-MS and GC-MS data, augmented by solid-state NMR and thioacidolysis, yields two types of new insights: the chemical compounds responsible for contrasting metabolic profiles of native and wound periderms, and the impact of FHT deficiency in each of these plant tissues. In the current report, we confirm a role for FHT in developing wound periderm and highlight its distinctive features as compared to the corresponding native potato periderm.

Transcription Factor MYB26 Is Key to Spatial Specificity in Anther Secondary Thickening Formation.

Successful fertilization relies on the production and effective release of viable pollen. Failure of anther opening (dehiscence), results in male sterility, although the pollen may be fully functional. MYB26 regulates the formation of secondary thickening in the anther endothecium, which is critical for anther dehiscence and fertility. Here, we show that although the MYB26 transcript shows expression in multiple floral organs, the MYB26 protein is localized specifically to the anther endothecium nuclei and that it directly regulates two NAC domain genes, NST1 and NST2, which are critical for the induction of secondary thickening biosynthesis genes. However, there is a complex relationship of regulation between these genes and MYB26. Using DEX-inducible MYB26 lines and overexpression in the various mutant backgrounds, we have shown that MYB26 up-regulates both NST1 and NST2 expression. Surprisingly normal thickening and fertility rescue does not occur in the absence of MYB26, even with constitutively induced NST1 and NST2, suggesting an additional essential role for MYB26 in this regulation. Combined overexpression of NST1 and NST2 in myb26 facilitates limited ectopic thickening in the anther epidermis, but not in the endothecium, and thus fails to rescue dehiscence. Therefore, by a series of regulatory controls through MYB26, NST1, NST2, secondary thickening is formed specifically within the endothecium; this specificity is essential for anther opening.

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation.

Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize.

Metal transport protein 8 in Camellia sinensis confers superior manganese tolerance when expressed in yeast and Arabidopsis thaliana.

Manganese (Mn) is an important micronutrient element required for plant growth and development, playing catalytic roles in enzymes, membranes and DNA replication. The tea plant (Camellia sinensis) is able to accumulate high concentration of Mn without showing signs of toxicity, but the molecular mechanisms underlying this remain largely unknown. In this study, the C. sinensis cultivar 'LJCY' had higher Mn tolerance than cultivar 'YS', because chlorophyll content reduction was lower under the high Mn treatment. Proteomic analysis of the leaves revealed that C. sinensis Metal Tolerance Protein 8 (CsMTP8) accumulated in response to Mn toxicity in cultivar 'LJCY'. The gene encoding CsMTP8, designated as CsMTP8 was also isolated, and its expression enhanced Mn tolerance in Saccharomyces cerevisiae. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana increased plant tolerance and reduced Mn accumulation in plant tissues under excess Mn conditions. Subcellular localization analysis of green florescence fused protein indicated that CsMTP8 was localized to the plasma membranes. Taken together, the results suggest that CsMTP8 is a Mn-specific transporter, which is localized in the plasma membrane, and transports excess Mn out of plant cells. The results also suggest that it is needed for Mn tolerance in shoots.

DEFECTIVE KERNEL1 (DEK1) Regulates Cell Walls in the Leaf Epidermis.

The plant epidermis is crucial to survival, regulating interactions with the environment and controlling plant growth. The phytocalpain DEFECTIVE KERNEL1 (DEK1) is a master regulator of epidermal differentiation and maintenance, acting upstream of epidermis-specific transcription factors, and is required for correct cell adhesion. It is currently unclear how changes in DEK1 lead to cellular defects in the epidermis and the pathways through which DEK1 acts. We have combined growth kinematic studies, cell wall analysis, and transcriptional analysis of genes downstream of DEK1 to determine the cause of phenotypic changes observed in DEK1-modulated lines of Arabidopsis (Arabidopsis thaliana). We reveal a novel role for DEK1 in the regulation of leaf epidermal cell wall structure. Lines with altered DEK1 activity have epidermis-specific changes in the thickness and polysaccharide composition of cell walls that likely underlie the loss of adhesion between epidermal cells in plants with reduced levels of DEK1 and changes in leaf shape and size in plants constitutively overexpressing the active CALPAIN domain of DEK1. Calpain-overexpressing plants also have increased levels of cellulose and pectins in epidermal cell walls, and this is correlated with the expression of several cell wall-related genes, linking transcriptional regulation downstream of DEK1 with cellular effects. These findings significantly advance our understanding of the role of the epidermal cell walls in growth regulation and establish a new role for DEK1 in pathways regulating epidermal cell wall deposition and remodeling.

Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities.

To date little has been done on identification of major phenolic compounds responsible for anticancer and antioxidant properties of pea (Pisum sativum L.) seed coat extracts. In the present study, phenolic profile of the seed coat extracts from 10 differently colored European varieties has been determined using ultrahigh-performance liquid chromatography-linear trap quadrupole orbitrap mass spectrometer technique. Extracts of dark colored varieties with high total phenolic content (up to 46.56 mg GAE/g) exhibited strong antioxidant activities (measured by 2,2-diphenyl-1-picrylhydrazyl or DPPH assay, and ferric ion reducing and ferrous ion chelating capacity assays) which could be attributed to presence of gallic acid, epigallocatechin, naringenin, and apigenin. The aqueous extracts of dark colored varieties exert concentration-dependent cytotoxic effects on all tested malignant cell lines (human colon adenocarcinoma LS174, human breast carcinoma MDA-MB-453, human lung carcinoma A594, and myelogenous leukemia K562). Correlation analysis revealed that intensities of cytotoxic activity of the extracts strongly correlated with contents of epigallocatechin and luteolin. Cell cycle analysis on LS174 cells in the presence of caspase-3 inhibitor points out that extracts may activate other cell death modalities besides caspase-3-dependent apoptosis. The study provides evidence that seed coat extracts of dark colored pea varieties might be used as potential cancer-chemopreventive and complementary agents in cancer therapy.

Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS.

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

Influence of berry ripeness on accumulation, composition and extractability of skin and seed flavonoids in cv. Sangiovese (Vitis vinifera L.).

BACKGROUND: The anthocyanin and tannin concentration and composition of Vitis vinifera L. cv. Sangiovese berries were investigated from post-veraison to harvest. Exhaustive extraction with methanol and acetone was performed to determine the total flavonoid concentration, while a model hydroalcoholic solution was used to prepare extracts representing the winemaking process. The aim of this study was to improve the knowledge of the phenolic maturity of Sangiovese grape. RESULTS: The total anthocyanin concentration increased during ripening, but the quantity of extractable anthocyanins increased more rapidly than the total. The total skin tannin concentration declined from post-veraison to harvest, whereas the extractable portion increased, with little difference in the composition of the fractions. Both the total and extractable seed tannin concentration diminished rapidly just after veraison, and only small fluctuations were detected until harvest. CONCLUSION: These results indicate that the extractability of anthocyanins and skin tannins increases during ripening, whereas there is no clear trend for seed tannins during the same period. This is the first survey to study the behavior of phenolic compounds during different steps of ripening of Sangiovese grape. © 2016 Society of Chemical Industry.

Epidermal jasmonate perception is sufficient for all aspects of jasmonate-mediated male fertility in Arabidopsis.

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1-YFP transgene in the coi1-1 mutant background. As COI1 is an essential component of the JA co-receptor complex, the null coi1-1 mutant is male sterile due to lack of JA perception. We show that expression of COI1-YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA-dependent non-autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.

Orchestration of three transporters and distinct vascular structures in node for intervascular transfer of silicon in rice.

Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.

Decrease in osmotically driven water flux and transport through mangrove roots after oil spills in the presence and absence of dispersants.

The objective of this study was to evaluate the effect of crude oil on water transport through mangroves roots in the presence and absence of dispersants. Water transport through the roots were evaluated experimentally using red mangrove root segments exposed to salt water contaminated with Louisiana crude oil for seven days in the presence and absence of Corexit 9500A (dispersant). Experimental observations were interpreted in view of the structural integrity and fouling phenomena observed on the epidermis and endodermis layers of the roots. The effects of oil on the radial water flux through the epidermis and endodermis were analyzed using a dual layer filtration model. Progression of fouling due to accumulation and penetration of the contaminants through the root layers were interpreted in relation to observed mangrove health (long and short term effects) reported in the literature.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. RESULTS: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. CONCLUSION: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

Cucumis sativus L. WAX2 Plays a Pivotal Role in Wax Biosynthesis, Influencing Pollen Fertility and Plant Biotic and Abiotic Stress Responses.

Cuticular waxes play an important part in protecting plant aerial organs from biotic and abiotic stresses. In previous studies, the biosynthetic pathway of cuticular waxes and relative functional genes has been researched and understood; however, little is known in cucumber (Cucumis sativus L.). In this study, we cloned and characterized an AtWAX2 homolog, CsWAX2, in cucumber and found that it is highly expressed in the epidermis, where waxes are synthesized, while subcellular localization showed that CsWAX2 protein is localized to the endoplasmic reticulum (ER). The transcriptional expression of CsWAX2 was found to be induced by low temperature, drought, salt stress and ABA, while the ectopic expression of CsWAX2 in an Arabidopsis wax2 mutant could partially complement the glossy stem phenotype. Abnormal expression of CsWAX2 in transgenic cucumbers specifically affected both very long chain (VLC) alkanes and cutin biosynthesis. Furthermore, transgenic cucumber plants of CsWAX2 showed significant changes in pollen viability and fruit resistance to water loss and pathogens compared with the wild type. Collectively, these results indicated that CsWAX2 plays a pivotal role in wax biosynthesis, influencing pollen fertility and the plant's response to biotic and abiotic stresses.

Transport Function of Rice Amino Acid Permeases (AAPs).

The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis.