The double knockdown of the mitochondrial uncoupling protein isoforms reveals partial redundant roles during Arabidopsis thaliana vegetative and reproductive development.

Mitochondrial uncoupling proteins (UCPs) are specialized proteins capable of dissipating the proton electrochemical gradient generated in respiration independent of ATP synthesis. Three UCP coding genes with distinct expression patterns have been identified in Arabidopsis thaliana (namely UCP1, UCP2 and UCP3). Here, we generated T-DNA double-insertion mutants (ucp1 ucp2, ucp1 ucp3 and ucp2 ucp3) to investigate the functionality of the Arabidopsis UCP isoforms. A strong compensatory effect of the wild-type UCP gene was found in the double-knockdown lines. Higher levels of reactive oxygen species (ROS) were observed in vegetative and reproductive organs of double mutant plants. This exacerbated oxidative stress in plants also increased lipid peroxidation but was not compensated by the activation of the antioxidant system. Alterations in O2 consumption and ADP/ATP ratio were also observed, suggesting a change in mitochondrial energy-generating processes. Deficiencies in double-mutants were not limited to mitochondria and also changed photosynthetic efficiency and redox state. Our results indicate that UCP2 and UCP3 have complementary function with UCP1 in plant reproductive and vegetative organ/tissues, as well as in stress adaptation. The partial redundancy between the UCP isoforms suggests that they could act separately or jointly on mitochondrial homeostasis during A. thaliana development.

Knockdown of Mitochondrial Uncoupling Proteins 1 and 2 (AtUCP1 and 2) in Arabidopsis thaliana Impacts Vegetative Development and Fertility.

Mitochondrial uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that dissipate the proton electrochemical gradient generated by the respiratory chain complexes. In plants, these proteins are crucial for maintaining mitochondrial reactive oxygen species (ROS) homeostasis. In this study, single T-DNA insertion mutants for two (AtUCP1 and AtUCP2) out of the three UCP genes present in Arabidopsis thaliana were employed to elucidate their potential roles in planta. Our data revealed a significant increase in the Adenosine triphosphate (ATP)/Adenosine diphosphate (ADP) ratios of both mutants, indicating clear alterations in energy metabolism, and a reduced respiratory rate in atucp2. Phenotypic characterization revealed that atucp1 and atucp2 plants displayed reduced primary root growth under normal and stressed conditions. Moreover, a reduced fertility phenotype was observed in both mutants, which exhibited an increased number of sterile siliques and a lower seed yield compared with wild-type plants. Reciprocal crosses demonstrated that both male fertility and female fertility were compromised in atucp1, while such effect was exclusively observed in the male counterpart in atucp2. Most strikingly, a pronounced accumulation of hydrogen peroxide in the reproductive organs was observed in all mutant lines, indicating a disturbance in ROS homeostasis of mutant flowers. Accordingly, the atucp1 and atucp2 mutants exhibited higher levels of ROS in pollen grains. Further, alternative oxidase 1a was highly induced in mutant flowers, while the expression profiles of transcription factors implicated in gene regulation during female and male reproductive organ/tissue development were perturbed. Overall, these data support the important role for AtUCP1 and AtUCP2 in flower oxidative homeostasis and overall plant fertility.

Identification of a seed maturation protein gene from Coffea arabica (CaSMP) and analysis of its promoter activity in tomato.

KEY MESSAGE: A seed maturation protein gene (CaSMP) from Coffea arabica is expressed in the endosperm of yellow/green fruits. The CaSMP promoter drives reporter expression in the seeds of immature tomato fruits. In this report, an expressed sequence tag-based approach was used to identify a seed-specific candidate gene for promoter isolation in Coffea arabica. The tissue-specific expression of the cognate gene (CaSMP), which encodes a yet uncharacterized coffee seed maturation protein, was validated by RT-qPCR. Additional expression analysis during coffee fruit development revealed higher levels of CaSMP transcript accumulation in the yellow/green phenological stage. Moreover, CaSMP was preferentially expressed in the endosperm and was down-regulated during water imbibition of the seeds. The presence of regulatory cis-elements known to be involved in seed- and endosperm-specific expression was observed in the CaSMP 5'-upstream region amplified by genome walking (GW). Additional histochemical analysis of transgenic tomato (cv. Micro-Tom) lines harboring the GW-amplified fragment (~ 1.4 kb) fused to uidA reporter gene confirmed promoter activity in the ovule of immature tomato fruits, while no activity was observed in the seeds of ripening fruits and in the other organs/tissues examined. These results indicate that the CaSMP promoter can be used to drive transgene expression in coffee beans and tomato seeds, thus representing a promising biotechnological tool.