A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants

Abstract

SummaryAntisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several 'antisense' potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32-43%, 56-71%, 79-83% and 91-97% in A-70, A-3, A-51 and A-2. Separation on a Q-Sepharose-FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose-1,7-biphosphatase and plastid fructose-1,6-bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A-70, A-3, A-51 and A-2. The transformants contained increased triose phosphates, and very low ribulose-1,5-bisphosphate and glycerate-3-phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A-70 and A-3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A-70, was decreased by 41% in A-3, and was severely inhibited in plants with under 20% of wild-type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.

Most of ADP·glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves

“…EC 2.7.7.27) is the only enzyme catalyzing the synthesis of ADPG, and it acts as the major limiting step of the gluconeogenic process (8)(9)(10). This popular view, consistent with the idea that the chloroplast is a complete photosynthetic unit that can produce starch from the atmospheric CO 2 (11,12), appears to be supported by a wide variety of mutant and transgenic lines in which enzymes and transport machineries have been modulated (13)(14)(15)(16)(17). However, there is a growing volume of evidence that points out inconsistencies with such mechanism and previews the likelihood of alternative gluconeogenic pathway(s) in which the ADPG linked to starch biosynthesis is produced in the cytosol (18)(19)(20).…”

“…As shown in Table 2, leaves from both control and 35S-ASPP- Leaf homogenates were prepared and subjected to centrifugation as described by Haake et al (16). Data are given as mean Ϯ SEM of three independent experiments.…”

“…Chloroplasts were prepared as described in Haake et al (16). The final pellet was resuspended in 50 mM Tris, pH 7.8͞5 mM MgCl 2 ͞1 mM EDTA͞2 mM DTT͞1% (vol/vol) Triton X-100.…”

Enzyme Activity Profiles during Fruit Development in Tomato Cultivars andSolanum pennellii

“…Fru-1,6-bisP aldolase was adapted from Haake et al (1998) and assayed in the direction of breaking the Fru-1,6-bisP down into glyceraldehyde 3-phosphate and DHAP. Extracts as well as DHAP standards, prepared in the extraction buffer and ranging from 0 to 1 nmol, were incubated in a solution containing 100 mM Tricine/KOH, pH 8.5, 5 mM MgCl 2 , 1 mM EDTA, 2 units mL -1 glycerol-3-phosphate dehydrogenase, 1 unit mL -1 TPI, 5 mM NADH, and 0 (blank) or 5 mM (maximal activity) Fru-1,6-bisP.…”

Transgenic Tobacco Plants Overexpressing Chloroplastic Ferredoxin-NADP(H) Reductase Display Normal Rates of Photosynthesis and Increased Tolerance to Oxidative Stress

“…As a consequence, decreases in protein levels caused by the introduction of antisense RNA in transgenic plants are partially compensated by further activation of the extant enzyme molecules, resulting in attenuation of the inhibitory effects. For example, rates of photosynthesis were not strongly affected until the content of Rubisco (Stitt et al, 1991;Hudson et al, 1992), aldolase (Haake et al, 1998), plastidic Fru-1,6-bisphosphatase (Kossmann et al, 1994), sedoheptulose-1,7-bisphosphatase (Harrison et al, 1998), and phosphoribulokinase (Paul et al, 1995) was reduced to 50% or less of their wild-type levels. Moreover, the effects usually became significant only under saturating CO 2 and illumination (Stitt et al, 1991).…”