Suppression of sucrose synthase affects auxin signaling and leaf morphology in tomato

<div><p>Metabolic enzymes have been found to play roles in plant development. Sucrose synthase (SUS) is one of the two enzyme families involved in sucrose cleavage in plants. In tomato, six SUS genes have been found. We generated transgenic tomato plants with RNAi suppression of <i>SlSUS1</i>, <i>SlSUS3</i> and <i>SlSUS4</i> genes. Independent transgenic lines with RNAi suppression of more than one <i>SUS</i> gene exhibited morphological effects on their cotyledons and leaf structure, but there were no significant effects on their carbohydrate levels, demonstrating that SUS has a developmental function, in addition to its metabolic function. Shoot apices of the transgenic lines showed elevated expression of <i>JAGGED</i> (<i>JAG</i>) and the auxin transporter <i>PIN1</i>. In a PIN1-GFP fusion reporter/SUS-RNAi hybrid, PIN1-GFP patterns were altered in developing leaves (as compared to control plants), indicating that <i>SlSUS</i> suppression alters auxin signaling. These results suggest possible roles for SUS in the regulation of plant growth and leaf morphology, in association with the auxin-signaling pathway.</p></div

Effect of <i>SlSUS</i> suppression on tomato fertility and seed viability.

<p><b>(A)</b><i>SlSUS-RNAi</i> and WT plants were grown in a greenhouse to maturity. Flowers from the first five inflorescences from the ground of each plant (<i>n</i> = 9) were counted. <b>(B)</b> Fruit set was calculated as the number of fruit divided by the number of flowers from the first five inflorescences of each plant (<i>n</i> = 9). <b>(C)</b> To calculate average seed weights, 50 seeds per fruit (<i>n</i> = 4) were counted and weighed, and that weight was divided by the number of seeds. Error bars represent the standard error. Asterisks indicate a statistically significant difference relative to the WT (* <i>P</i> < 0.05; ** <i>P</i> < 0.01).</p

The S1R4 line exhibits abnormal leaf morphology.

<p><b>(A)</b> Mature S1R4 leaf, <b>(B)</b> mature WT leaf, <b>(C)</b> magnification of the boxed areas in the S1R4 leaf, <b>(D)</b> magnification of the boxed areas in the WT leaf and <b>(E)</b> close-ups of the adaxial and <b>(F)</b> the abaxial side of an S1R4 leaf, showing ectopic blade formation of the leaflets (red area). Note the angle of the petiolules (arrowheads) and of the leaflet curling (arrow). Bar– 2 cm.</p

<i>SlSUS-RNAi</i> lines exhibit abnormal cotyledon morphology.

<p><b>(A)</b> Cotyledons from wild-type (WT) seedlings and seedlings of three <i>SlSUS-RNAi</i> lines. Each pair of cotyledons was taken from a single seedling. Bar– 1 cm. <b>(B)</b> S1R4 embryo and <b>(C)</b> WT embryo extracted from seeds soaked for 24 h. The warped cotyledon of the S1R4 embryo is clearly visible; bar– 0.5 mm.</p

Expression patterns of the three <i>SlSUS</i> genes in reproductive organs.

<p>Comparative GUS stains of flowers and fruit from tomato lines expressing GUS under the control of each of the three <i>SlSUS</i> promoters (<i>proSlSUS</i>). Leftmost column: flowers before anthesis; second column from left: flowers at anthesis; third column from left: ~1 cm green fruit; rightmost column: ripe fruit; bar– 2 mm.</p

<i>NtAQP1</i> complements growth inhibition of <i>AtHXK1</i>.

<p>(A) Representative images of 5-week-old tomato plants homozygous for <i>NtAQP1</i> (AQP1), <i>AtHXK1</i> (HK4) or both genes (AQP1xHK4). (B) Height (<i>n</i>≥8) and (C) leaf area (<i>n</i>≥6) of 9-week-old plants. (D) Hexokinase activity was determined using protein extracted from mature leaves of WT, AQP1, HK4 and AQP1xHK4 plants. Data are means of five independent biological repeats ± SE. (E) Relative expression of <i>SlCAB1</i> (<i>Solanum lycopersicum</i> a/b binding protein) in WT, AQP1, HK4 and AQP1xHK4 plants. Data are means of five-six independent biological repeats ± SE. (B–E) Different letters indicate a significant difference (<i>t</i> test, <i>P</i><0.05).</p

Transpiration rate of AQP1xHK4 plants.

<p>The rate of transpiration was monitored continuously throughout the day for each line (AQP1, HK4, AQP1xHK4 and WT). The presented data are the means ± SE for each 10^th sampling point (<i>n</i> = 6). The transpiration data were normalized to the total leaf area and the amount taken up by the neighboring submerged fixed-size wick each day, which was set to 100%.</p

Photosynthetic and hydraulic characteristics of WT, AQP1, AQP1xHK4 and HK4 plants.

<p><i>L</i>_r, root hydraulic conductance; <i>K</i>_sx, xylem-specific stem hydraulic conductivity; <i>A</i>_N, net photosynthesis; <i>g</i>_s, stomatal conductance; <i>g</i>_m, mesophyll CO₂ conductance; <i>C</i>_i, substomatal CO₂ concentration; C_c, Chloroplast CO₂ concentration; <i>J</i>, the rate of electron transport. Presented data are means ± SE (<i>n</i>, number of replicates, as indicated in parentheses). Different letters in a row indicate significant differences (<i>t</i> test, <i>P</i><0.05).</p