MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking.

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-delta tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.

CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis.

To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix((R)) ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana nectaries. One of the genes displaying highly enriched expression in nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars.

Uncovering the Arabidopsis thaliana nectary transcriptome: investigation of differential gene expression in floral nectariferous tissues.

BACKGROUND: Many flowering plants attract pollinators by offering a reward of floral nectar. Remarkably, the molecular events involved in the development of nectaries, the organs that produce nectar, as well as the synthesis and secretion of nectar itself, are poorly understood. Indeed, to date, no genes have been shown to directly affect the de novo production or quality of floral nectar. To address this gap in knowledge, the ATH1 Affymetrix GeneChip array was used to systematically investigate the Arabidopsis nectary transcriptome to identify genes and pathways potentially involved in nectar production. RESULTS: In this study, we identified a large number of genes differentially expressed between secretory lateral nectaries and non-secretory median nectary tissues, as well as between mature lateral nectaries (post-anthesis) and immature lateral nectaries (pre-anthesis). Expression within nectaries was also compared to thirteen non-nectary reference tissues, from which 270 genes were identified as being significantly upregulated in nectaries. The expression patterns of 14 nectary-enriched genes were also confirmed via RT PCR. Upon looking into functional groups of upregulated genes, pathways involved in gene regulation, carbohydrate metabolism, and lipid metabolism were particularly enriched in nectaries versus reference tissues. CONCLUSION: A large number of genes preferentially expressed in nectaries, as well as between nectary types and developmental stages, were identified. Several hypotheses relating to mechanisms of nectar production and regulation thereof are proposed, and provide a starting point for reverse genetics approaches to determine molecular mechanisms underlying nectar synthesis and secretion.

Arabidopsis thaliana as a model for functional nectary analysis.

Nectaries and nectar have received much research attention for well over 200 years due to their central roles in plant-pollinator interactions. Despite this, only a few genes have demonstrated impacts on nectary development, and none have been reported to mediate de novo nectar production. This scarcity of information is largely due to the lack of a model that combines sizeable nectaries, and high levels of nectar production, along with suitable genomics resources. For example, even though Arabidopsis thaliana has been useful for developmental studies, it has been largely overlooked as a model for studying nectary function due to the small size of its flowers. However, Arabidopsis nectaries, along with those of related species, are quite operational and can be used to discern molecular mechanisms of nectary form and function. A current understanding of the machinery underlying nectary function in plants is briefly presented, with emphasis placed on the prospects of using Arabidopsis as a model for studying these processes.

Identification of differential gene expression in Brassica rapa nectaries through expressed sequence tag analysis.

BACKGROUND: Nectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa. METHODOLOGY AND PRINCIPAL FINDINGS: Four cDNA libraries were created using RNA isolated from the median and lateral nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5' end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes. CONCLUSIONS/SIGNIFICANCE: This work provided a snapshot of gene expression in actively secreting B. rapa nectaries, and also allowed the identification of differential gene expression between median and lateral nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa.

Identification, cloning and characterization of a GDSL lipase secreted into the nectar of Jacaranda mimosifolia.

The presence and function of several proteins secreted into floral nectars has been described in recent years. Here we report the presence of at least eight distinct proteins secreted into the floral nectar of the tropical tree Jacaranda mimosifolia (Bignoniaceae). Steps were initiated to identify and characterize these proteins in order to determine potential functions. The N-terminal sequence of the major Jacaranda nectar protein, JNP1, at 43 kDa contained similarity with members of the plant GDSL lipase/esterase gene family. Based upon this sequence, a full-length cDNA was isolated and predicted to encode a mature protein of 339 amino acids with a molecular mass of 37 kDa. Both raw nectar and heterologously expressed JNP1 displayed lipase/esterase activities. Interestingly, J. mimosifolia flowers produce an opaque, white colored nectar containing spherical, lipophilic particles approximately 5 microm in diameter and smaller. GS-MS analysis also identified the accumulation of free fatty acids within the nectar. It is proposed that JNP1 hydrolyzes Jacaranda nectar lipids with the concomitant release of free fatty acids. Potential functions of JNP1 in relation to pollinator attraction and prevention of microbial growth within nectar are briefly discussed.