SUMMARYAuxin regulates plant growth and development in part by activating gene expression. Arabidopsis thaliana SMALL AUXIN UP RNAs (SAURs) are a family of early auxin-responsive genes with unknown functionality. Here, we show that transgenic plant lines expressing artificial microRNA constructs (aMIR-SAUR-A or -B) that target a SAUR subfamily (SAUR61-SAUR68 and SAUR75) had slightly reduced hypocotyl and stamen filament elongation. In contrast, transgenic plants expressing SAUR63:GFP or SAUR63:GUS fusions had long hypocotyls, petals and stamen filaments, suggesting that these protein fusions caused a gain of function. SAUR63:GFP and SAUR63:GUS seedlings also accumulated a higher level of basipetally transported auxin in the hypocotyl than did wild-type seedlings, and had wavy hypocotyls and twisted inflorescence stems. Mutations in auxin efflux carriers could partially suppress some SAUR63:GUS phenotypes. In contrast, SAUR63:HA plants had wild-type elongation and auxin transport. SAUR63:GFP protein had a longer half-life than SAUR63:HA. Fluorescence imaging and microsomal fractionation studies revealed that SAUR63:GFP was localized mainly in the plasma membrane, whereas SAUR63:HA was present in both soluble and membrane fractions. Low light conditions increased SAUR63:HA protein turnover rate. These results indicate that membrane-associated Arabidopsis SAUR63 promotes auxin-stimulated organ elongation.

Adventitious roots emerge from aerial plant tissues, and the induction of these roots is essential for clonal propagation of agriculturally important plant species. This process has received extensive study in horticultural species but much less focus in genetically tractable model species. We have explored the role of auxin transport in this process in Arabidopsis (Arabidopsis thaliana) seedlings in which adventitious root initiation was induced by excising roots from low-light-grown hypocotyls. Inhibition of auxin transport from the shoot apex abolishes adventitious root formation under these conditions. Root excision was accompanied by a rapid increase in radioactive indole-3-acetic acid (IAA) transport and its accumulation in the hypocotyl above the point of excision where adventitious roots emerge. Local increases in auxin-responsive gene expression were also observed above the site of excision using three auxin-responsive reporters. These changes in auxin accumulation preceded cell division events, monitored by a cyclin B1 reporter (pCYCB1;1:GUS), and adventitious root initiation. We examined excisioninduced adventitious root formation in auxin influx and efflux mutants, including auxin insensitive1, pin-formed1 (pin1), pin2, pin3, and pin7, with the most profound reductions observed in ATP-binding cassette B19 (ABCB19). An ABCB19 overexpression line forms more adventitious roots than the wild type in intact seedlings. Examination of transcriptional and translational fusions between ABCB19 and green fluorescent protein indicates that excision locally induced the accumulation of ABCB19 transcript and protein that is temporally and spatially linked to local IAA accumulation leading to adventitious root formation. These experiments are consistent with localized synthesis of ABCB19 protein after hypocotyl excision leads to enhanced IAA transport and local IAA accumulation driving adventitious root formation.

This study utilized tomato (Solanum lycopersicum) mutants with altered flavonoid biosynthesis to understand the impact of these metabolites on root development. The mutant anthocyanin reduced (are) has a mutation in the gene encoding FLAVONOID 3-HYDROXYLASE (F3H), the first step in flavonol synthesis, and accumulates higher concentrations of the F3H substrate, naringenin, and lower levels of the downstream products kaempferol, quercetin, myricetin, and anthocyanins, than the wild type. Complementation of are with the p35S:F3H transgene reduced naringenin and increased flavonols to wild-type levels. The initiation of lateral roots is reduced in are, and p35S:F3H complementation restores wild-type root formation. The flavonoid mutant anthocyanin without has a defect in the gene encoding DIHYDROFLAVONOL REDUCTASE, resulting in elevated flavonols and the absence of anthocyanins and displays increased lateral root formation. These results are consistent with a positive role of flavonols in lateral root formation. The are mutant has increased indole-3-acetic acid transport and greater sensitivity to the inhibitory effect of the auxin transport inhibitor naphthylphthalamic acid on lateral root formation. Expression of the auxin-induced reporter (DR5-b-glucuronidase) is reduced in initiating lateral roots and increased in primary root tips of are. Levels of reactive oxygen species are elevated in are root epidermal tissues and root hairs, and are forms more root hairs, consistent with a role of flavonols as antioxidants that modulate root hair formation. Together, these experiments identify positive roles of flavonols in the formation of lateral roots and negative roles in the formation of root hairs through the modulation of auxin transport and reactive oxygen species, respectively.

Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis.

The branched-chain amino acid transaminases (BCATs) have a crucial role in metabolism of the branched-chain amino acids leucine, isoleucine, and valine. These enzymes catalyze the last step of synthesis and the initial step of degradation of these amino acids. Although the biosynthetic pathways of branched chain amino acids in plants have been extensively investigated and a number of genes have been characterized, their catabolism in plants is not yet completely understood. We previously characterized the branched chain amino acid transaminase gene family in tomato, revealing both the subcellular localization and kinetic properties of the enzymes encoded by six genes. Here, we examined possible functions of the enzymes during fruit development. We further characterized transgenic plants differing in the expression of branched chain amino acid transaminases 1 and 3, evaluating the rates of respiration in fruits deficient in BCAT1 and the levels of volatiles in lines overexpressing either BCAT1 or BCAT3. We quantitatively tested, via precursor and isotope feeding experiments, the importance of the branched chain amino acids and their corresponding keto acids in the formation of fruit volatiles. Our results not only demonstrate for the first time the importance of branched chain amino acids in fruit respiration, but also reveal that keto acids, rather than amino acids, are the likely precursors for the branched chain flavor volatiles.

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