Rosemary J. Birch

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis.

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2(AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1−) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3antisense plants. In keeping with the mild phenotype compared with Rsw1−, reductions in total cellulose levels in antisenseCesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, likeCesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 inrsw1 but were unable to complement that mutants defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31°C.

Morphology of rsw1, a cellulose deficient mutant of Arabidopsis thaliana

SummaryTherswl mutant ofArabidopsis thaliana is mutated in a gene encoding a cellulose synthase catalytic subunit. Mutant seedlings produce almost as much cellulose as the wild type at 21 °C but only about half as much as the wild type at 31 °C. We used this conditional phenotype to investigate how reduced cellulose production affects growth and morphogenesis in various parts of the plant. Roots swell in all tissues at 31 °C, and temperature changes can repeatedly switch them between swollen and slender growth patterns. Dark-grown hypocotyls also swell, whereas cotyledons and rosette leaf blades are smaller, their surfaces are more irregular and their petioles shorter. Leaf trichomes swell and branch abnormally. Plants readily initiate inflorescences at 31 °C which have shorter but not fatter bolts and stomata which bulge above the uneven surface of internodes. Bolts carry the normal number of flowers, but their stigmas protrude beyond the shortened sepals and petals. Anthers dehisce normally, but self-fertilisation is reduced because the stigma is well above the anthers. Anther filaments are short and show a crumpled surface. Viable pollen develops, but female reproductive competence and postpollination development are severely impaired. We conclude that theRSW1 gene is important for cellulose synthesis in many parts of the plant and that reduced cellulose synthesis suppresses organ expansion rather than organ initiation, causes radial swelling only in the root and hypocotyl, but makes the surfaces of many organs uneven. We discuss some possible reasons to explain why different organs vary in their responses. The morphological changes suggest that RSW1 contributes cellulose to primary walls but do not yet exclude a role during secondary-wall deposition.

Arabidopsis dynamin-like protein DRP1A: a null mutant with widespread defects in endocytosis, cellulose synthesis, cytokinesis, and cell expansion

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis.

Chimeric Proteins Suggest That the Catalytic and/or C-Terminal Domains Give CesA1 and CesA3 Access to Their Specific Sites in the Cellulose Synthase of Primary Walls

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3WT protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3rsw5) does not.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Significance Using a translational photosynthesis approach, we successfully increased CO2-assimilation in leaf chloroplasts of the model plant tobacco. Phylogenetic analysis revealed parallel evolutionary linkages between the large (L-) subunit of the CO2-fixing enzyme Rubisco and its molecular chaperone Rubisco accumulation factor 1 (RAF1). We experimentally tested and exploited this correlation using plastome transformation, producing plants that demonstrated the role of RAF1 in L-subunit assembly and resolve the RAF1 quaternary structure as a dimer. We show the increase in Rubisco biogenesis translated to improvements in leaf photosynthesis and growth of the plants. The outcomes have application to the growing interest into identifying and implementing strategies to supercharge photosynthesis to improve crop productivity and stem global food-security concerns. Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco’s biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tobAtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tobAtL-R1 plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tobAtL-R1 and Arabidopsis leaves at 10–15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8AS8t Rubisco [comprising AtL and tobacco small (S) subunits] in tobAtL-R1 leaves compared with tobAtL, despite >threefold lower steady-state Rubisco mRNA levels in tobAtL-R1. Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tobAtL-R1 lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.

Infant perception of the shapes of tilted plane forms

This paper describes three experiments based on Caron, Caron, and Carlson (1979) . In each case, 3-month-old infants were familiarized with a plane form presented at various tilts. In three subsequent test trials they saw the familiar form in a familiar (60°) orientation, the familiar form in a novel frontoparallel orientation (novel-orientation test), and a novel form in frontoparallel (novel-form test). The novel form was the frontoparallel projection of the familiar form when tilted at 60°. In Experiment 1, recovery of fixation occurred in the novel-form test when the infants were familiarized with a rectangle and the novel form was a trapezoid. No recovery occurred when both the familiar and novel forms were irregular quadrilaterals. The results for the rectangle were interpreted as evidence of shape constancy. The results for the irregular forms were attributed to a poor resolution of these forms. In Experiment 2, the infants were familiarized with a rectangle as in Experiment 1. However, the novel-orientation test stimulus was reduced in size so that its area equalled that of the trapezoid seen in the novel-form test (novel-size test). While a recovery of fixation occurred in the novel-form test, no recovery occurred to the reduction of size in the novel-size test. This was taken as evidence that the rectangle/trapezoid discrimination was based on the perception of form rather than size. In Experiment 3, the procedure of Experiment 1 was repeated but the infants were familiarized with either a parallelogram or a trapezoid. In neither case was there a recovery in the novel-form test. It was concluded that the rectangle/trapezoid discrimination was based upon a perception of orthogonality.