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. 2018 May;177(1):369-380.
doi: 10.1104/pp.17.01417. Epub 2018 Mar 19.

Heterologous Expression of AtBBX21 Enhances the Rate of Photosynthesis and Alleviates Photoinhibition in Solanumtuberosum

Carlos D Crocco  1 , Gabriel Gomez Ocampo  1 , Edmundo L Ploschuk  2 , Anita Mantese  3 , Javier F Botto  4
Affiliations

Heterologous Expression of AtBBX21 Enhances the Rate of Photosynthesis and Alleviates Photoinhibition in Solanumtuberosum

Carlos D Crocco et al. Plant Physiol. 2018 May.

Abstract

B-box (BBX) proteins are zinc-finger transcription factors containing one or two B-box motifs. BBX proteins act as key factors in the networks regulating growth and development. The relevance of BBX21 to light and abscisic acid signaling in seedling development is well established; however, its importance in adult plant development and agronomic species is poorly understood. Therefore, we studied the effect of heterologous expression of Arabidopsis (Arabidopsis thaliana) BBX21 in potato (Solanum tuberosum) var Spunta. Three independent AtBBX21-expressing lines and the wild-type control were cultivated under sunlight and at controlled temperatures in a greenhouse. By anatomical, physiological, biochemical, and gene expression analysis, we demonstrated that AtBBX21-expressing plants were more robust and produced more tubers than wild-type plants. Interestingly, AtBBX21-expressing plants had higher rates of photosynthesis, with a significant increase in photosynthetic gene expression, and higher stomatal conductance, with increased size of the stomatal opening, without any associated decline in water use efficiency. Furthermore, AtBBX21-expressing potato plants had reduced photoinhibition associated with higher production of anthocyanins and phenolic compounds, and higher expression of genes in the phenylpropanoid biosynthesis pathway. To gain insights into the mechanism of BBX21, we evaluated the molecular, morphological, metabolic, and photosynthetic behavior in adult BBX21-overexpressing Arabidopsis. We conclude that BBX21 overexpression improved morphological and physiological attributes, and photosynthetic rates in nonoptimal, high-irradiance conditions, without associated impairment of water use efficiency. These characteristics of BBX21 may be useful for increasing production of potatoes, and potentially of other crops.

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Figures

Figure 1.
Figure 1.
Transgenic AtBBX21 potato lines are more robust and shorter than nontransformed plants. A, Representative photograph of 28-d-old nontransformed (Spunta) and transgenic potato plants (CH1, CH2, and CH13). B, AtBBX21 transcript levels of the three independent transgenic lines used in this study (n = 10). C, Plant height and stem diameter in 21-d-old plants (n = 10). D, Fresh weight relative to dry weight in 35-d-old plants (n = 10). n = number of biological replicates. Mean values are shown with error bars indicating se. Asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 2.
Figure 2.
Heterologous expression of AtBBX21 promotes stem vasculature and leaf thickening in potato plants. A, Cross section of apex, middle, and basal stems of nontransformed (Spunta) and transgenic plants (CH2). B, Layer number of parenchymal cells (n = 3). C, Leaf lamina width (n = 3). p, Parenchyma; v, vascular bundle; pm, palisade mesophyll cell; sm, spongy mesophyll cells. Bar = 50 µm. n = technical replicates for each genotype. Mean values are shown with error bars indicating se. Asterisks indicate significant difference between nontransformed and transgenic lines (**P < 0.01 and ***P < 0.001).
Figure 3.
Figure 3.
Heterologous expression of AtBBX21 increases tuber yield in potato plants. A, Representative photographs of nontransformed (Spunta) and transgenic tubers (CH2) at the end of the experiment. B, Tuber weight, tuber number (n = 12), and starch content (n = 5). C, Amyloplast length, width, and number (n = 3). D, Cross section of tubers. a, Amyloplast. Bar = 10 µm. n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant).
Figure 4.
Figure 4.
Heterologous expression of AtBBX21 increases photosynthesis in potato plants. A, Chlorophyll levels of nontransformed (Spunta) and transgenic (CH1, CH2, and CH13) potato leaves (n = 10). B, Photosynthesis, transpiration rate, stomatal conductance, and internal concentration of CO2 as a function of PPFD (n = 4). C, WUE as function of PPFD (n = 4). D, StRCA, StLHCB, and StFTSZ1 transcript levels (n = 4). n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant).
Figure 5.
Figure 5.
Heterologous expression of AtBBX21 increases stomatal aperture in potato leaves. A, Stomatal density and stomatal index estimated as the number of abaxial stomata/adaxial stomata of nontransformed (Spunta) and transgenic (CH2) potato leaves (n = 3). B, Stomatal aperture (n = 3). C, Representative photographs of stomata in nontransformed and BBX21-overexpressing lines. S, Stomata; O, opercule. Bar = 10 µm. n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05 and ***P < 0.001; NS, not significant).
Figure 6.
Figure 6.
Heterologous expression of AtBBX21 increases anthocyanin and phenolic levels in potato leaves. A, Phenolics and anthocyanins in nontransformed (Spunta) and transgenic (CH1, CH2, and CH13) potato leaves (n = 5). B, Sunscreen pigment accumulation in young and totally expanded leaves. Sunscreen accumulation is revealed by a decrease in the intensity of UV-induced chlorophyll fluorescence (less fluorescence indicates higher accumulation of anthocyanin and phenolic compounds). C, Maximal photochemical efficiency (Fv/Fm) in 14-d-old plants acclimated at low irradiance (200 μmol m−2 s−1) and then exposed at high irradiance (900 μmol m−2 s−1) for 2 h (n = 6). n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05 and **P < 0.01; NS, not significant).
Figure 7.
Figure 7.
Heterologous expression of AtBBX21 in potato promotes gene expression and metabolites accumulation related with the phenylpropanoid pathway in leaves. A, Diagram of phenylpropanoid pathway and key enzymes. B, PAL, CHS, CHI, and F3H transcript levels (n = 4). C, Chlorogenic acid 1, chlorogenic acid 2, and quercetin glycoside metabolites measured by HPLC (n = 5). n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between nontransformed and transgenic lines (*P < 0.05; NS, not significant).
Figure 8.
Figure 8.
BBX21 overexpression promotes rosette expansion, branching, and accumulation of anthocyanins and phenolics in Arabidopsis. A, Representative photographs showing vegetative rosette (35-d-old plants, left) and secondary flowering ramifications (98-d-old plants, right) in wild-type (Col) and BBX21-overexpresed (BBX21-OE) Arabidopsis plants. B, Leaf length, width of the fourth expanded leaf, and number of secondary flowering axes (n = 8). C, Anthocyanin and phenolic content in leaves (n = 5). D, CHS, CHI, and F3H transcript levels (n = 3). n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between Col and BBX21-OE Arabidopsis plants (*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 9.
Figure 9.
BBX21 overexpression promotes photosynthesis in Arabidopsis. A, Photosynthesis, transpiration rate, stomatal conductance, and internal concentration of CO2 as function of PPFD (n = 4). B, WUE as function of PPFD (n = 4). C, FD1, RCA, and LHCA transcripts (n = 3). n = number of biological replicates. Mean values are shown with error bars indicating se. Data were analyzed by Student’s t tests, and asterisks indicate significant difference between Col and BBX21-OE plants (*P < 0.05 and **P < 0.01; NS, not significant).
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Comment in

  • Improving on the Humble Spud.
    Hayes S. Hayes S. Plant Physiol. 2018 May;177(1):5-6. doi: 10.1104/pp.18.00350. Plant Physiol. 2018. PMID: 29720531 Free PMC article. No abstract available.

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