Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH 2 to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

SummaryATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxi®cation processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic ®brosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.

Narrow leaflet soybean (Glycine max) varieties tend to have more seeds per pod than broad leaflet varieties. Narrow leaflet in soybean is conferred by a single recessive gene, ln. Here, we show that the transition from broad (Ln) to narrow leaflet (ln) is associated with an amino acid substitution in the EAR motif encoded by a gene (designated Gm-JAGGED1) homologous to Arabidopsis JAGGED (JAG) that regulates lateral organ development and the variant exerts a pleiotropic effect on fruit patterning. The genomic region that regulates both the traits was mapped to a 12.6-kb region containing only one gene, Gm-JAG1. Introducing the Gm-JAG1 allele into a loss-of-function Arabidopsis jagged mutant partially restored the wild-type JAG phenotypes, including leaf shape, flower opening, and fruit shape, but the Gm-jag1 (ln) and EAR-deleted Gm-JAG1 alleles in the jagged mutant did not result in an apparent phenotypic change. These observations indicate that despite some degree of functional change of Gm-JAG1 due to the divergence from Arabidopsis JAG, Gm-JAG1 complemented the functions of JAG in Arabidopsis thaliana. However, the Gm-JAG1 homoeolog, Gm-JAG2, appears to be sub-or neofunctionalized, as revealed by the differential expression of the two genes in multiple plant tissues, a complementation test, and an allelic analysis at both loci.

SummaryPreviously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P 2 , decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P 2 modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P 2 -binding domain GFP:PLCd1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P 2 increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P 2 substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P 2 targets we performed patchclamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P 2 . Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P 2 in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P 2 . The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P 2 in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.

No abstract