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The three amino acid loop extension (TALE) homeodomain superfamily, which comprises the KNOTTED-like and BEL1-like families, plays a critical role in regulating meristem activity. We previously demonstrated a function for KNAT6 (for KNOTTED-like from Arabidopsis thaliana 6) in shoot apical meristem and boundary maintenance during embryogenesis. KNAT2, the gene most closely related to KNAT6, does not play such a role. To investigate the contribution of KNAT6 and KNAT2 to inflorescence development, we examined their interactions with two TALE genes that regulate internode patterning, BREVIPEDICELLUS (BP) and PENNYWISE (PNY). Our data revealed distinct and overlapping interactions of KNAT6 and KNAT2 during inflorescence development. Removal of KNAT6 activity suppressed the pny phenotype and partially rescued the bp phenotype. Removal of KNAT2 activity had an effect only in the absence of both BP and KNAT6 or in the absence of both BP and PNY. Consistent with this, KNAT6 and KNAT2 expression patterns were enlarged in both bp and pny mutants. Thus, the defects seen in pny and bp are attributable mainly to the misexpression of KNAT6 and to a lesser extent of KNAT2. Hence, our data showed that BP and PNY restrict KNAT6 and KNAT2 expression to promote correct inflorescence development. This interaction was also revealed in the carpel.

The homeobox gene family plays a crucial role during the development of multicellular organisms. The KNOTTED-like genes from Arabidopsis thaliana (KNAT6 and KNAT2) are close relatives of the meristematic genes SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS, but their function is not currently known. To investigate their role, we identified null alleles of KNAT6 and KNAT2. We demonstrate that KNAT6 contributes redundantly with STM to the maintenance of the shoot apical meristem (SAM) and organ separation. Consistent with this role, the expression domain of KNAT6 in the SAM marks the boundaries between the SAM and cotyledons. The lack of meristematic activity in the knat6 stm-2 double mutant and the fusion of cotyledons were linked to the modulation of CUP-SHAPED COTYLEDON (CUC) activity. During embryogenesis, KNAT6 is expressed later than STM and CUC. In agreement with this fact, CUC1 and CUC2 were redundantly required for KNAT6 expression. These data provide the basis for a model in which KNAT6 contributes to SAM maintenance and boundary establishment in the embryo via the STM/CUC pathway. KNAT2, although the closest related member of the family to KNAT6, did not have such a function.

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the alpha,beta-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the alpha,beta-unsaturated bond of the photoproduced reactive carbonyls.

Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.

Using a transgenic line that overexpresses a fusion of the KNAT2 (KNOTTED-like Arabidopsis) homeodomain protein and the hormone-binding domain of the glucocorticoid receptor (GR), we have investigated the possible relations between KNAT2 and various hormones. Upon activation of the KNAT2-GR fusion, we observed a delayed senescence of the leaves and a higher rate of shoot initiation, two processes that are also induced by cytokinins and inhibited by ethylene. Furthermore, the activation of the KNAT2-GR fusion induced lobing of the leaves. This feature was partially suppressed by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, or by the constitutive ethylene response ctr1 mutation. Conversely, some phenotypic traits of the ctr1 mutant were suppressed by the activation of the KNAT2-GR fusion. These data suggest that KNAT2 acts synergistically with cytokinins and antagonistically with ethylene. In the shoot apical meristem, the KNAT2 gene is expressed in the L3 layer and the rib zone. 1-Aminocyclopropane-1-carboxylic acid treatment restricted the KNAT2 expression domain in the shoot apical meristem and reduced the number of cells in the L3. The latter effect was suppressed by the activation of the KNAT2-GR construct. Conversely, the KNAT2 gene expression domain was enlarged in the ethylene-resistant etr1-1 mutant or in response to cytokinin treatment. These data suggest that ethylene and cytokinins act antagonistically in the meristem via KNAT2 to regulate the meristem activity.

gamma-Glutamyl transpeptidase (gamma-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the gamma-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for gamma-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound gamma-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis gamma-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Budding yeast strains that produced the Arabidopsis thaliana protein CEF or its amino-terminal proline-rich domain were more tolerant to hydroperoxides. CEF is homologous to animal and yeast Sec24 proteins. These data suggest that CEF plays a protective role through protein transport during growth under pro-oxidant conditions.

The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M. & Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was > 1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed.