Sucrose export defective1 encodes a novel protein implicated in chloroplast-to-nucleus signaling

The Sucrose export defective1 (Sxd1) gene of maize was cloned and shown to encode a novel protein conserved between plants and cyanobacteria. The structure of the Sxd1 locus was determined in wild-type plants and two independent sxd1 alleles. Expression analysis demonstrated that the gene was transcribed in all green tissues, with highest levels in maturing leaf blades. In situ hybridization studies revealed high levels of Sxd1 mRNA in bundle sheath cells, with lower levels within the mesophyll. The SXD1 protein was localized to chloroplasts, in both bundle sheath and mesophyll cells. Levels of sucrose, glucose, and fructose were compared between wild-type and sxd1 plants. Mutant plants were fully capable of producing sucrose and accumulated all three sugars at concentrations above those measured in wild-type plants. Despite these increased sugar concentrations, photosynthetic gene expression was not significantly downregulated in affected areas of sxd1 leaf blades. These results are consistent with photosynthate being trapped within anthocyanin-accumulating regions of sxd1 leaves due to plasmodesmal occlusion at the bundle sheath-vascular parenchyma boundary of the minor veins. A model for SXD1 function is proposed in which the protein is involved in a chloroplast-to-nucleus signaling pathway necessary for proper late-stage differentiation of maize bundle sheath cells, including the developmentally regulated modification of plasmodesmata.     

Effects of moderate drought on ascorbate peroxidase and glutathione reductase activities in mesophyll and bundle sheath cells of maize

Mesophyll and bundle sheath cells of maize leaves ( Zea mays L.) both contain the enzymes ascorbate peroxidase (AP; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) which are involved in hydrogen peroxide detoxification. Since bundle sheath cells of maize are deficient in photosystem II and have high CO₂ levels, oxidative stress may be less severe in these cells than in mesophyll cells. The present study was conducted to determine if AP and GR activity levels preferentially increase in mesophyll cells relative to bundle sheath cells when plants are subjected to moderate drought. Although drought inhibited the growth of greenhouse-grown plants, it did not affect the levels of protein, chlorophyll or AP. GR was unaffected by drought in whole leaf tissue and mesophyll cells, but did increase slightly in bundle sheath cells. This slight increase is of questionable biological importance. AP and GR activity levels were similar in mesophyll cells, bundle sheath cells and in whole leaf tissue. The data suggest that moderate drought has little effect on enzymes of the hydrogen peroxide scavenging system and that mesophyll and bundle sheath cells may be exposed to similar levels of hydrogen peroxide.     

Transgenic maize lines with cell‐type specific expression of fluorescent proteins in plastids

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll‐specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath‐specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon‐optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid‐related studies of wild‐type and mutant maize plants and provide material from which different plastid types may be isolated.     

Low temperature-induced changes in the distribution of H2O2 and antioxidants between the bundle sheath and mesophyll cells of maize leaves

The distribution of antioxidants between bundle sheath and mesophyll cells of maize leaves was analysed in plants grown at 20 degrees C, 18 degrees C and 15 degrees C. The purity of the isolated bundle sheath and mesophyll fractions was determined using compartment-specific marker enzymes. In plants grown at 15 degrees C, ascorbate peroxidase, CuZn-superoxide dismutase (CuZn-SOD) and monodehydroascorbate reductase activities were increased in the bundle sheath cells, and glutathione reductase, dehydroascorbate reductase and monodehydroascorbate reductase activities were enhanced in the mesophyll cells. SOD was absent from the mesophyll of plants grown at 20 degrees C but an Fe-SOD activity was found in the mesophyll of plants grown at 15 degrees C. Foliar Mn-SOD activities were decreased at 15 degrees C compared to 20 degrees C. Catalase was undetectable in the mesophyll extracts of plants grown at 15 degrees C. Ascorbate and glutathione contents were considerably higher in the mesophyll than the bundle sheath fractions of plants grown at 20 degrees C. The ratios of reduced to oxidized forms of these antioxidants were significantly decreased in the bundle sheath, but increased in the mesophyll of leaves grown at 15 degrees C. Foliar H2O2 accumulated at 15 degrees C compared to 20 degrees C. Most of the foliar H2O2 was localized in the mesophyll tissues at all growth temperatures. The differential distribution of antioxidants between leaf bundle sheath and mesophyll tissues, observed at 20 degrees C, is even more pronounced when plants are grown at 15 degrees C and may contribute to the extreme sensitivity of maize to low temperatures.     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Different pH-dependences of K+ channel activity in bundle sheath and mesophyll cells of maize leaves

The isolation of bundle sheath protoplasts from leaves of Zea mays L. for patch clamp whole-cell experiments presents special problems caused by the suberin layer surrounding these cells. These problems were overcome by the isolation technique described here. Two different types of whole-cell response were found: a small response caused by MB-1 (maize bundle sheath conductance type 1) which was instantaneously activated, and another caused by MB-2 (maize bundle sheath conductance type 2) consisting of an instantaneous response (maize bundle sheath K⁺ instantaneous current type 2; MB-KI2) similar to but stronger than the current through MB-1 plus a small time-dependent outward rectifying component (maize bundle sheath activated outward rectifying current; MB-AOR) with voltage-dependent delayed activation. The occurrence of MB-AOR was often accompanied by a smaller contribution from an inward rectifying channel at negative potentials. Activation of MB-2 required ATP. It is suggested that MB-1 and MB-2 are related to bundle sheath cells with and without direct contact with the xylem vessels. In mesophyll cells, only one type of response caused by MM-2 (maize mesophyll conductance type 2) was found with an instantaneous (maize mesophyll K⁺ instantaneous current type 2, MM-KI2) and a voltage-dependent delayed component (maize mesophyll activated outward rectifying current, MM-AOR). The most striking difference between bundle sheath and mesophyll cells was the pH dependence of K⁺ uptake. At pH 7.2, uptake of K⁺ by MB-2 was identical to that by MM-2 over the whole voltage range. However, acidification stimulated K⁺ conductance in bundle sheath cells, whereas a decrease was found for MM-2. At pH 6.15, the bundle sheath channel MB-2 had more than a 10-fold higher K⁺ uptake at positive and negative potentials than MM-2. The channel MB-1, too, was stimulated by low pH. This seems to indicate a putative role for MB-1 and MB-2 in charge balance during uptake of nutrients via cotransport from the xylem into the symplasm. Received: 23 April 1999 / Accepted: 19 July 1999     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.