Availability of Rubisco small subunit up-regulates the transcript levels of large subunit for stoichiometric assembly of its holoenzyme in rice

Abstract

Rubisco is composed of eight small subunits coded for by the nuclear RBCS multigene family and eight large subunits coded for by the rbcL gene in the plastome. For synthesis of the Rubisco holoenzyme, both genes need to be expressed coordinately. To investigate this molecular mechanism, the protein synthesis of two subunits of Rubisco was characterized in transgenic rice (Oryza sativa) plants with overexpression or antisense suppression of the RBCS gene. Total RBCS and rbcL messenger RNA (mRNA) levels and RBCS and RbcL synthesis simultaneously increased in RBCS-sense plants, although the increase in total RBCS mRNA level was greater. In RBCS-antisense plants, the levels of these mRNAs and the synthesis of the corresponding proteins declined to a similar extent. The amount of RBCS synthesized was tightly correlated with rbcL mRNA level among genotypes but not associated with changes in mRNA levels of other major chloroplast-encoded photosynthetic genes. The level of rbcL mRNA, in turn, was tightly correlated with the amount of RbcL synthesized, the molar ratio of RBCS synthesis to RbcL synthesis being identical irrespective of genotype. Polysome loading of rbcL mRNA was not changed. These results demonstrate that the availability of RBCS protein up-regulates the gene expression of rbcL primarily at the transcript level in a quantitative manner for stoichiometric assembly of Rubisco holoenzyme.

Figure 1.

Protein synthesis and the mRNA levels of Rubisco. Protein synthesis of RBCS (A) and RbcL (B) and transcript levels of RBCS (C) and rbcL (D) were determined in expanding young leaves of wild-type (WT), RBCS-sense (26-8 and 35-4), and RBCS-antisense (AS-71) plants. Values are expressed per unit of leaf fresh weight (FW). Data are presented as means ± se (n = 3). Asterisks indicate statistically significant differences from the values of wild-type plants by Dunnett’s test (P < 0.05). In C, white, light gray, dark gray, and black columns represent transcript levels of RBCS2, -3, -4, and -5, respectively.

Figure 2.

Relationships between protein synthesis and transcript levels of Rubisco. A, RBCS synthesis versus total RBCS mRNA level. B, RbcL synthesis versus rbcL mRNA level. Circles, squares, diamonds, and triangles represent the wild-type plant (WT), RBCS-sense line 26-8, RBCS-sense line 35-4, and RBCS-antisense line AS-71, respectively. Data are taken from Figure 1. A dotted line is drawn through the origin and data from wild-type plants. FW, Fresh weight.

Figure 3.

Polysome loading of Rubisco mRNAs. Transcript levels of each fraction were expressed as percentages of the sum of all fractions. A to E show the data of RBCS2, -3, -4, and -5 and rbcL, respectively. F shows the distribution of total RNA among the fractions. Symbols are the same as in Figure 2. Asterisks indicate statistically significant differences from the values of wild-type plants (WT) by Dunnett’s test (P < 0.05).

Figure 4.

Relationships between RBCS and rbcL. A, rbcL mRNA level versus RBCS synthesis. B, rbcL mRNA level versus total RBCS mRNA level. Data are taken from Figure 1. Symbols and lines are the same as in Figure 2. FW, Fresh weight, WT, wild type.

Figure 5.

Relationship between mRNA levels of other chloroplast photosynthetic components and RBCS synthesis. A to D show the results of petA, atpB, psbA, and psaA, respectively. These transcript levels are expressed as relative values where the data from the wild-type plants (WT) are defined as 1. Symbols are the same as in Figure 2. Data are presented as means ± se (n = 3). FW, Fresh weight.