Influence of starch deficiency on photosynthetic and post-photosynthetic carbon isotope fractionations.

Carbon isotope (13C) fractionations occurring during and after photosynthetic CO2 fixation shape the carbon isotope composition (δ13C) of plant material and respired CO2. However, responses of 13C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ13C of organic matter (δ13COM), concentrations and δ13C of potential respiratory substrates, δ13C of dark-respired CO2 (δ13CR), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ13C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ13COM, δ13CR, and δ13C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ13C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic 13C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ13C variations in starch-deficient mutants are primarily explained by photosynthetic 13C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

Light promotion of hypocotyl gravitropism of a starch-deficient tobacco mutant correlates with plastid enlargement and sedimentation.

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.

Severely reduced gravitropism in dark-grown hypocotyls of a starch-deficient mutant of Nicotiana sylvestris.

Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10 degrees for NS 458 and about 70 degrees for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.

Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris.

Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.