ABS3 Subfamily Of MATE Transporters Regulate Hypocotyl Cell Elongation And Leaf Senescence In Arabidopsis

The growth and development of higher plants is under complex regulations to ensure the elaboration of developmental programs under changing environment. To dissect these regulatory circuits, we have carried out genetic screens for abnormal shoot(abs) mutants with altered shoot development programs in Arabidopsis thaliana.Here, we report the isolation of two semi-dominant mutants, abs3-1D and abs4-1D, through activation tagging screens in Arabidopsis. Both mutants showed a dwarf and “bushy” phenotype. We determined that ABS3 and ABS4 code for two closely related putative Multidrug and Toxic Compound Extrusion(MATE) family of efflux transporters, respectively. Moreover, over-expressions of ABS3 and ABS4,as well as two related MATE genes ABS3-Like1(ABS3L1) and ABS3L2, showed similar effects on Arabidopsis development. In this thesis the functions of ABS3, ABS4, ABS3L1 and ABS3L2 in cell elongation and leaf senescenc were investigated.(1) Through large-scale screens for mutants that displayed altered shoot development programs, two abnormal shoot(abs) mutants abs3-D and abs4-1D, were identified. Both mutants showed thephenotypesof more compact stature, shortened petioles, an increased rate of leaf initiation and lateral branch development. We confirmed that increased transcriptions of two MATE transporter genes ABS3 and ABS4 are the causes for mutant phenotypes, respectively. The over-expressions of ABS3L1 and ABSL2, which are closely related toABS3 and ABS4, can lead to similar phenotypes.These results indicate that increased expressions of ABS3 subfamily of MATE transporter genes including ABS3, ABS4, ABS3L1 and ABS3L2 are capable of altering shoot developmental programs in a similar manner.(2)By tissue specific semi-quantitative RT-PCRs and GUS stainings of promoter-GUS transgenic plants, we demonstrated that ABS3, ABS4, ABS3L1 and ABS3L2 have distinctive tissue and developmental expression profiles. The expressions of ABS3 subfamily of MATE genes are highly regulated spatially and temporally.(3) Through protoplast transient transformation and co-localization analysis, ABS3, ABS4, ABS3L1 and ABS3L2 were confirmed to have highly similar subcellular localizations, and they all localized to the late endosome/prevacuole(LE/PVC) compartment of the endomembrane system.(4)ABS3, ABS4, ABS3L1 and ABS3L2 loss-of-function mutants were obtained. However, the overall growth and development was not grossly altered in any of the single knockout mutant compared to wild type. Higher order mutant of these MATE genes was generated, and abs3-1abs4-1abs3l1-1abs3l2-1 quadruple mutant(referred to as mateq hereafter) showed a more pronounced developmental phenotype.(5) Arabidopsis hypocotyl system was used for dissecting the function of ABS3 subfamily MATE transporters during cell elongation.Over-expression of ABS3 and ABS4 individually led to the inhibition of hypocotyl cell elongation in the light. On the other hand, mateq showed the opposite phenotype of an enhanced hypocotyl cell elongation in the light. These data indicate that ABS3 subfamilyMATE genes negatively regulate hypocotyl cell elongation in light-grown seedlings.(6) Hypocotyl cell elongation and de-etiolation processes in the dark were also affected by the mutations of ABS3 subfamily MATE genes.Over-expression of these four genes individually led to the inhibition of hypocotyl cell elongation in the dark. Exogenously applied sucrose attenuated the inhibition of hypocotyl elongation caused by abs3-1D and abs4-1D in the dark, and enhanced the hypocotyl elongation of mateq under prolonged dark treatment. These data demonstrate that over-production of ABS3 and ABS3-LikeMATE genes is capable of negatively regulating cell elongation in the dark.(7) A suppressor mutant F07-08 was isolated by screening for genetic modifiers of abs3-1D. F07-08 single mutant was then confirmed as a new mutant allele of photoreceptor gene PHYTOCHROME B(PHYB).F07-08 single mutant can also suppress the phenotype of abs4-1D. These data suggest a genetic interaction between ABS3, ABS4 and PHYB.(8) Under the dark treatment, abs3-1D showed leaf early senescence phenotype and mateq showed the opposite phenotype of stay-green. By using individually darkened leaf treatment, we found that the darkened leaves of asb3-1D showed accelerated leaf senescence, with the chlorophyll content, photosynthesis efficiency, protein content decreased faster than wildtype. Moreover, Senescence Associate Genes(SAG) were induced earlier in individually darkened abs3-1D leaves. On the contrary, individually darkened leaves of mateq showed stay-green phenotypes. Overexpression of ABS3 gene accelerates leaf senescence under darktreatment, and the absence of ABS3 subfamily MATE genes can delay leaf senescence.(9) Nutrient starvation can also trigger premature leaf senescence.abs3-1Dshowed early leaf senescence phenoyptesunder C-starvation and N-starvation conditions, while mateq showed stay-green phenotypes. Thus, ABS3 subfamilyof MATE genes are also involved in nutrient starvation related leaf senescence regulation.