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Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein–protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. BR perception at the plasma membrane initiates a series of phosphorylation events enabling the nuclear accumulation and activity of the key transcription factors BZR1 and BES1. Previous studies support that all BR signaling components form an interconnected pathway, although it is not known how these proteins are brought together for the prompt signal transduction from the plasma membrane to the nucleus. Here we report that plant-specific Tetratricopeptide Thioredoxin- Like (TTL) proteins are positive regulators of BR signaling that function as scaffold for the BR signaling components in Arabidopsis. TTL3 directly interacts with BZR1 and the phosphatase BSU1, and associates in vivo with most core components involved in BR signaling, with the exception of the BAK1 coreceptor. TTL3 is mainly localized in the cytoplasm, and BR treatment increases its localization at the plasma membrane. In addition, the expression of TTL3 strengthens the association of BR-signaling components BSK1 and BZR1 at the plasma membrane. Consistent with a role in BR signaling, mutations in TTL3, and related TTL1 and TTL4 genes cause reduced BR responses, and these defects that highly enhanced in a triple ttl1 ttl3 ttl4 mutant. We propose a novel mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex with the goal of ensuring dephosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1. Interestingly, this novel TTL scaffold model for BR signaling resembles that of Wnt signaling in metazoans, in which TTL proteins would act similar to Axin1, optimizing signaling efficiency of the cascade by promoting the assembly of the signaling complex at the plasma membrane.

We are developing molecular approaches to study the growth and development of woody plants. As part of our research efforts, we report the molecular cloning and characterization of PsRLK here, a cDNA from the conifer Scots pine (Pinus sylvestris L.) encoding a polypeptide similar to the receptor protein kinases described in angiosperms. A full-length clone was isolated from a cDNA library constructed with poly (A)+ enriched RNA prepared from germinating pine seeds. Characterization of the isolated sequence revealed that it contains multiple leucine-rich repeats in the N-terminal region and a characteristic Ser/Thr protein kinase domain in the C-terminal region. N- and C-terminal conserved domains are separated by a putative membrane spanning sequence. PsRLK protein is encoded by a single gene in the pine genome. A comparison of the pine sequence with the LRR-RLKs from Arabidopsis revealed that PsRLK is phylogenetically related to the LRR XI subfamily members. RT-PCR analyses of transcript abundance in pine tissues suggest that the gene expression pattern of PsRLK reflects the plant body formation programme, with increased levels during development of pine seedlings. The precise localization of PsRLK transcripts revealed that gene expression was restricted to specialized phloem cells suggesting a possible function of the putative receptor-like protein kinase in this particular vascular element.

Changes in the expression of cytosolic glutamine synthetase (GS1; EC 6.3, 1.2) and chloroplastic glutamine synthetase (GS2) were examined in tomato (Lycopersicon esculentum Mill.) leaves during natural senescence. Total GS activity during this process declined to 4% of the highest value reached at the time of maximum leaf growth rate. Changes of activity were probably caused by a decrease in the amount of GS2 polypeptides. The presence of GS1 polypeptides was evident during the late senescence stage of the leaf despite the low content of total soluble protein. The differential expression of GS genes during leaf senescence in tomato plants was followed using gene-specific homologous probes synthesized from cloned cDNAs encoding cytosolic and chloroplastic GS polypeptides. Northern blot analysis showed that the level of GS2 transcripts decreased during leaf senescence as did the amounts of GS2 polypeptide, but messengers for GS2 were undetectable in the very late senescence stage when a small amount of polypeptides was still present. The content of mRNA detected with the 3’non-coding region of the GS1 probe dramatically increased with leaf senescence, thus explaining the presence of GS1 polypeptides at this stage, although there was no correspondence between the amount of GS protein and the high abundancy of GS1 mRNA. The presence of both GS1 polypeptide and messenger in the leaf, and their increase during natural leaf senescence, support a role for GS1 in this process.

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experiment approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzene-tricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.