A conserved KIN17 curved DNA-binding domain protein assembles with SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 to adapt Arabidopsis growth and development to limiting copper availability

Abstract

Proper copper (Cu) homeostasis is required by living organisms to maintain essential cellular functions. In the model plant Arabidopsis (Arabidopsis thaliana), the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor participates in reprogramming global gene expression during Cu insufficiency in order to improve the metal uptake and prioritize its distribution to Cu proteins of major importance. As a consequence, spl7 null mutants show morphological and physiological disorders during Cu-limited growth, resulting in lower fresh weight, reduced root elongation, and chlorosis. On the other hand, the Arabidopsis KIN17 homolog belongs to a well-conserved family of essential eukaryotic nuclear proteins known to be stress activated and involved in DNA and possibly RNA metabolism in mammals. In the study presented here, we uncovered that Arabidopsis KIN17 participates in promoting the Cu deficiency response by means of a direct interaction with SPL7. Moreover, the double mutant kin17-1 spl7-2 displays an enhanced Cu-dependent phenotype involving growth arrest, oxidative stress, floral bud abortion, and pollen inviability. Taken together, the data presented here provide evidence for SPL7 and KIN17 protein interaction as a point of convergence in response to both Cu deficiency and oxidative stress.

Figure 1.

KIN17 physically interacts with SPL7. A, KIN17 coimmunoprecipitates with SPL7. Total proteins from tobacco leaves transiently expressing the indicated tagged proteins were extracted (input) as described in “Materials and Methods” and pulled down by means of an anti-HA antibody (bound). Input and bound fractions were then assayed by western blot with anti-GFP-HRP antibodies to test for SPL7 coimmunoprecipitation. Membrane reprobed with an anti-HA-HRP is shown as a control for the pull down. B, BiFC assay for the nYFP::SPL7 and cYFP::KIN17 interaction. Epidermal cells of coinfiltrated tobacco leaves were examined after 3 d for restoration of YFP fluorescence using laser confocal microscopy. The YFP and chlorophyll signals are shown in color-separated and merged images. Bars = 100 µm (top row) and 10 µm (bottom row).

Figure 2.

KIN17 and SPL7 proteins localize to the nucleus in tobacco leaves. The entire KIN17 and SPL7 coding sequences were N-terminal fused to GFP and transiently expressed in tobacco leaves. Samples were observed using laser confocal microscopy 3 d post infiltration. The GFP fluorescence signal and the transmitted light (bright field) of representative samples are shown in separated and merged images. Bar = 10 µm.

Figure 3.

Characterization of the KIN17 spatial expression pattern. A to F, Histochemical GUS staining in pKIN17::GUS lines. A and B, Five-day-old (A) and 7-d-old (B) seedlings. C, Root vasculature. D, Rosette leaves. E, Postanthesis flowers. F, Anthers. G, KIN17 and SPL7 distribute mainly on aerial organs. Total RNA from seedlings and different adult plant organs, seedlings (Se), roots (R), rosette leaves (RL), cauline leaves (CL), stems (St), flowers (Flo), and siliques (Sil), was isolated and reverse transcribed to cDNA. KIN17 and SPL7 mRNA relative amounts were measured by qPCR with specific oligonucleotides and normalized to ACTIN1 and ELONGATION FACTOR1α levels. Error bars correspond to the sd of technical replicates (n = 3).

Figure 4.

Isolation of the kin17-1 mutant line. A, Scheme representing the KIN17 gene and the translated protein with its conserved domains defined. The T-DNA insertion in the kin17-1 line is indicated by an inverted triangle and corresponding flanking sequences. The positions of the oligonucleotides used to genotype the lines are shown by arrows (F, I, and R). The Zn finger domain (ZFD), Kin17, and KOW domains are indicated with squares. aa, Amino acids. B, Discrimination of homozygous kin17-1 plants. kin17-1 plants were self-crossed, and the offspring was genotyped with the oligonucleotide combinations indicated in A: for the wild-type (WT) allele, F + R; for the insertion allele, F + I. C, The kin17-1 line is a knockdown mutant. Total RNA from 6-d-old seedlings of the wild type, kin17-1, and pKIN17::KIN17 in the kin17-1 mutant background grown on 1/2 MS medium supplemented with Suc and BCS was prepared and reverse transcribed to cDNA. KIN17 mRNA relative levels were determined by qPCR and normalized to ACTIN1 and ELONGATION FACTOR1α levels. Error bars correspond to sd values of at least three independent biological replicates. The asterisk indicates a statistically significant difference from the wild type (P < 0.05).

Figure 5.

Deregulation of selected SPL7 targets in the kin17-1 mutant. Total RNA from 7-d-old seedlings of the wild type (WT), kin17-1, and pKIN17::KIN17 in the kin17-1 mutant background grown on 1/2 MS medium supplemented with Suc and 50 µm BCS was prepared and reverse transcribed to cDNA. The relative mRNA levels of the indicated targets were determined by qPCR and normalized to ACTIN1 and ELONGATION FACTOR1α transcripts. Error bars correspond to sd values of at least three independent biological replicates. Asterisks indicate statistically significant differences from the wild type (P < 0.05).

Figure 6.

Double mutant kin17-1 spl7-2 seedlings exhibit hypersensitivity to Cu deprivation. A, Ten-day-old seedlings of the wild type (WT), kin17-1, spl7-2, and kin17-1 spl7-2 germinated and grown on 1/2 MS supplemented with Suc and 5 µm CuSO₄ (+Cu) or 50 µm BCS (–Cu). B, The same four lines were grown on vertical plates under identical conditions for 6 d. Three representative seedlings of each line and treatment were photographed. C, Measurement of main root lengths of seedlings as grown and shown in B. Error bars indicate sd (n ≥ 10 biological replicates). Asterisks indicate a statistically significant difference from the wild type (P < 0.001).

Figure 7.

Phenotypes of the double mutant kin17-1 spl7-2 grown on soil. A and B, Representative 6-week-old wild-type (WT), kin17-1, spl7-2, and kin17-1 spl7-2 double mutant plants cultivated under long-day conditions on standard soil (A) or on soil treated with CuSO₄ (+Cu; B). C to E, Detailed macro images of inflorescences of selected kin17-1 spl7-2 plants grown on non-Cu-supplemented soil as shown in A.

Figure 8.

Measurement of oxidative stress-related parameters. A, The anthocyanin content is increased in kin17-1 spl7-2 double mutants when grown on standard soils. Anthocyanins from 1-month-old rosette leaves were extracted and quantified as described in “Materials and Methods” and normalized against fresh weight (FW). B, Lipid peroxidation is increased in kin17-1 spl7-2 grown on standard soils. Malondialdehyde (MDA) amounts in 1-month-old rosette leaves from the lines indicated were determined as described in “Materials and Methods” and normalized against fresh weight. Error bars correspond to the sd of at least six independent biological samples. Asterisks indicate statistically significant differences from the wild type (WT; *P < 0.05, ***P < 0.001).

Figure 9.

Determination of pollen production and viability. A, Pollen production in kin17-1 spl7-2 double mutant anthers is reduced compared with the wild type (WT) when grown on standard soil. Pollen was extracted from pooled anthers dissected from flowers just before anthesis in positions 5 to 10 of main inflorescences of plants cultivated as shown in Figure 7. Error bars indicate sd values of six countings representing 30 anthers in total per genotype. B, Impact of different Cu regimes on pollen viability. Pollen from recently opened flowers was immersed in a drop of FDA-Suc solution, and the percentage of fluorescent pollen was scored. Error bars represent sd values of at least four independent biological experiments. Asterisks indicate statistically significant differences in comparison with the wild type (*P < 0.05, ***P < 0.001).

Figure 10.

A working model for the KIN17-SPL7 node function under Cu deficiency. ROS are constantly generated in plant cells, especially due to PETC activity. Increased levels of ROS cause DNA damage and eventually result in abrogation of cell proliferation. The availability of Cu determines which enzymatic antioxidant response plants activate to counteract the deleterious effect of ROS. Whereas Cu is used to fulfill Cu-dependent antioxidant responses led by the Cu/Zn SOD during Cu sufficiency, it must be economized for fundamentally important Cu proteins during Cu-deficient periods. Consequently, the SPL7 transcription factor promotes a Cu-uptake and -redistribution strategy to preferentially allocate Cu toward PC and thus maintain the PETC as functional. Additionally, SPL7 also coordinates the Cu/Zn SOD substitution by the Fe SOD isoform to ensure a Cu-independent antioxidant response. In the case of high ROS levels, we propose that Arabidopsis KIN17 acts at three levels to counteract this scenario: (1) KIN17 would associate with SPL7 either to enhance the Cu-starvation response and reinforce the cellular antioxidant system or to protect additional yet-unknown Cu-dependent processes related to plant growth and development; (2) KIN17 would converge, directly or indirectly, with SPL7 to maintain some Cu-independent antioxidant system; and (3) KIN17 would also be directly involved in repairing DNA lesions, as reported in mammals.