Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplastenriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N-and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.
Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.
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® 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.
Plants offer metabolically rich floral nectar to attract visiting pollinators. The composition of nectar includes not only sugars, but also amino acids. We have examined the amino acid content of the nectar of ornamental tobacco and found that it is extremely rich (2 mM) in proline. Because insect pollinators preferentially utilize proline during the initial phases of insect flight and can reportedly taste proline, we determined whether honeybees showed a preference for synthetic nectars rich in proline. We therefore established an insect preference test and found that honeybees indeed prefer nectars rich in the amino acid proline. To determine whether this was a general phenomenon, we also examined the nectars of two insect-pollinated wild perennial species of soybean. These species also showed high levels of proline in their nectars demonstrating that plants often produce proline-rich floral nectar. Because insects such as honeybees prefer proline-rich nectars, we hypothesize that some plants offer proline-rich nectars as a mechanism to attract visiting pollinators.
Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-␥ (VPE␥) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPE␥ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpe␥ mutants but not in WT plants, suggesting a unique role of VPE␥ in protein processing and degradation in Arabidopsis. Many vacuolar enzymes are synthesized as pro-proteins and need to be proteolytically processed to be active. Plantspecific, endoplasmic reticulum (ER)-derived compartments containing precursors of cysteine proteinases (PPVs) have been described in seed-storage tissues (1-4). Germination of the seeds induces the expression and processing of those proteases into the mature active forms, which are thought to participate in the degradation of cellular materials in senescing storage tissues, presumably to assist in the programmed cell death process and to provide nutrients to the growing embryo. However, direct evidence linking these compartments with the senescence process in plants is lacking, which in part may be because seed PPVs have been found in legumes, which are not very amenable to genetic studies. Recently, similar compartments also called the ER bodies have been described in vegetative tissues of Arabidopsis (2). In this article we refer to these compartments in Arabidopsis vegetative tissues as PPVs.Two cysteine proteinases, vacuolar processing enzyme-␥ (VPE␥) and RD21, are known to be present in Arabidopsis PPVs. For RD21, it has been shown that PPVs store the inactive precursor isoform. Senescence induces the expression of VPE␥ and RD21 and the processing of RD21 into the mature active form (5), most likely in the acidic environment of the vacuole. In daylily, PPVs containing cysteine proteinase have been found in petals, which in this species undergo rapid senescence within 24 h of flower opening. These observations indicate that vegetative PPVs may also have a senescence-associated function, as has been suggested for seed PPVs. VPE␥ belongs to the small gene family of VPEs (6) that are thought to process storage proteins in vacuoles of developing seeds. Processing activity of castor bean and pumpkin VPE on several seed-storage proteins has been demonstrated in vitro (7,8). By analogy, the Arabidopsis seed-specific VPE isoform, VPE, is thought to process seedstorage proteins. In contrast, the substrates of VPE␥, which is expressed in vegetative tissues, are not known. However, through heterologous expression in yeast it has been shown that VPE␥ processes the vacuolar pro-protein precursor of yeast carboxypeptidase Y (CPY), indicating that VPE␥ has vacuolar processing activity.We ...
SUMMARYThe PIN family of proteins is best known for its involvement in polar auxin transport and tropic responses. PIN6 (At1g77110) is one of the remaining PIN family members in Arabidopsis thaliana to which a biological function has not yet been ascribed. Here we report that PIN6 is a nectary-enriched gene whose expression level is positively correlated with total nectar production in Arabidopsis, and whose function is required for the proper development of short stamens. PIN6 accumulates in internal membranes consistent with the ER, and multiple lines of evidence demonstrate that PIN6 is required for auxin-dependent responses in nectaries. Wild-type plants expressing auxin-responsive DR5:GFP or DR5:GUS reporters displayed intense signal in lateral nectaries, but pin6 lateral nectaries showed little or no signal for these reporters. Further, exogenous auxin treatment increased nectar production more than tenfold in wild-type plants, but nectar production was not increased in pin6 mutants when treated with auxin. Conversely, the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) reduced nectar production in wild-type plants by more than twofold, but had no significant effect on pin6 lines. Interestingly, a MYB57 transcription factor mutant, myb57-2, closely phenocopied the loss-of-function mutant pin6-2. However, PIN6 expression was not dependent on MYB57, and RNA-seq analyses of pin6-2 and myb57-2 mutant nectaries showed little overlap in terms of differentially expressed genes. Cumulatively, these results demonstrate that PIN6 is required for proper auxin response and nectary function in Arabidopsis. These results also identify auxin as an important factor in the regulation of nectar production, and implicate short stamens in the maturation of lateral nectaries.
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