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Cellular Differentiation In Plants And Other Essays For Scholarships

The Bradyrhizobium diazoefficiens USDA110 blr7537-encoded protein is a BclA homologue

Similar to other analyzed Bradyrhizobium strains9, no bacA homologous gene was identified in the genome of B. diazoefficiens USDA110. However, the B. diazoefficiens gene blr7537 was identified as a homolog of the bclA genes of Bradyrhizobium spp. ORS285 and ORS2789. The encoded protein has 70–72% identity and 80–81% similarity to the BclA proteins of strains ORS285 and ORS278. The protein has the same structure with an N-terminal SbmA-BacA transmembrane domain and a C-terminal ATPase domain. It thus encodes a potentially functional ABC transporter. Moreover, the genes in the 3 Bradyrhizobium species are located in syntenic regions that extend to over 200 kb. Therefore we designated blr7537 as bclA. Similarly as in strains ORS285 and ORS278, the B. diazoefficiens locus lacks genes that potentially encode additional components of the ABC transporter, such as a periplasmic binding protein for substrate binding and delivery to the transporter.

Bradyrhizobium diazoefficiens USDA110 BclA is an NCR peptide transporter and functional in symbiosis

To test the in vitro and in vivo activity of the B. diazoefficiens USDA110 BclA protein, a deletion mutant of the bclA gene was constructed. The gene was also cloned into the plasmids pMG103 and pRF771, downstream of the trp promoter, and introduced into the bclA, bacA and sbmA mutants of Bradyrhizobium sp. ORS285, S. meliloti strain Sm1021 and E. coli strain BW25113, respectively.

Bleomycin and Bac7 are antimicrobial compounds which have intracellular targets, DNA and the ribosomes respectively, and they require active transport to be taken up in the bacterial cells. In E. coli, S. meliloti and Bradyrhizobium spp. ORS285 and ORS278, the uptake is mediated by the SbmA/BacA/BclA transporters9, 17, 30. Thus strains expressing one of these transporters display a significantly increased sensitivity to bleomycin or Bac7. We find that wild type B. diazoefficiens strain USDA110 or the E. coli sbmA and S. meliloti bacA mutants expressing USDA110 bclA from the pRF771 plasmid are more sensitive to bleomycin or Bac7 than the corresponding strains lacking bclA (Fig. 1), in agreement with BclA of USDA110 being able to transport these peptides.

Contrary to bleomycin and Bac7, the sensitivity to antimicrobial NCR peptides is reduced in the presence of sbmA, bacA or bclA8, 9. This opposite response is likely because the toxicity of the NCR peptides resides in their potential to provoke membrane permeability and loss of membrane potential, rather than in the inhibition of some intracellular process. Also the bclA gene of USDA110 is able to reduce the sensitivity to NCR peptides in the S. meliloti bacA mutant (Fig. 2a). In addition, similarly as shown before for the S. meliloti bacA or Bradyrhizobium strain ORS285 bclA genes9, the expression of USDA110 bclA promotes the uptake of an FITC-modified NCR247 peptide into the S. meliloti strain Sm1021ΔbacA (Fig. 2b), while FITC alone is not taken up (Fig. 2c).

Finally, the bclA gene of USDA110 can complement the ORS285ΔbclA mutant for bacteroid differentiation in A. indica nodules (Fig. 3a–f). This mutant induces small nodules in which bacteria do not differentiate and die as revealed by live/dead staining of nodule sections9 (Fig. 3c,d). The USDA110 bclA gene, similarly as the ORS285 bclA gene, and introduced into this mutant on the pMG103 plasmid, restores the wild type phenotype with the formation of large nodules inhabited with well-formed spherical bacteroids that remain alive (Fig. 3a,b,e,f). Plants inoculated with both complemented strains showed vigorous growth, contrary to those inoculated with the un-complemented mutant indicating that nitrogen fixation defect of the mutant is restored by the USDA110 bclA gene. Moreover, the USDA110 bclA gene also complements in part the Sm1021ΔbacA strain for nodulation of M. sativa (Fig. 3g–l). The nodules induced by the complemented strain become elongated and pinkish, compared to the small white nodules induced by the bacA mutant (Fig. 3g,i,k). The bacA mutant bacteria die as soon as they are released inside the nodule cells and exposed to the NCR peptides8(Fig. 3j). The bacteroids of the complemented strain however are viable within the symbiotic cells indicating that the hypersensitivity of the bacA mutant to the NCR peptides is suppressed by the bclA gene of USDA110 (Fig. 3l). Nevertheless, the defect of the S. meliloti bacA mutation is only partially repaired by bclA of USDA110 because nodules do not fix nitrogen and do not support plant growth (data not shown). This phenotype is similar to the one obtained with the S. meliloti bacA complementation by bclA of strain ORS2859 or even by more similar bacA genes of Sinorhizobium or Rhizobium species31. This suggests that although rhizobial bacA and bclA genes have overlapping specificity for peptide uptake, they may also display differences in the set and/or amount of NCR peptides they can handle, probably because they evolved in the context of specific interactions with host plants, each producing its specific arsenal of NCR peptides. Nevertheless, the USDA110 bclA gene seems to be capable to treat the Aeschynomene NCR peptides.

Together, the bleomycin, Bac7 and NCR peptide sensitivity assays as well as the in vivo complementation of the Bradyrhizobium sp. ORS285 bclA mutantion or the S. meliloti bacA mutation indicate that the bclA gene of B. diazoefficiens strain USDA110 is functional and has a similar activity as the bclA gene of Bradyrhizobium strain ORS285 and the S. meliloti bacA gene.

Bradyrhizobium diazoefficiens USDA110 BclA is not required for symbiosis with NCR-lacking soybean

In agreement with the taxonomic position of soybean within the Millettioids, the B. diazoefficiens strain USDA110 bacteroids in soybean nodules are undifferentiated and display no cell enlargement and polyploidy as revealed by microscopy of nodule sections (Fig. 4c) and flow cytometry analysis of purified bacteroids (Fig. 4e). We tested whether the bclA gene in strain USDA110 is required for symbiosis with soybean. The bclA mutant of USDA110 was undistinguishable from the wild type strain for all parameters analyzed, including nodule tissue structure and bacterial occupation, bacterial viability, morphology, size and DNA content as well as nitrogen fixation (Fig. 4a–e). Thus the Bradyrhizobium bclA gene, similarly to bacA in other rhizobium species, is not required for symbiosis when bacteroids are not constrained by the host plant-produced NCR peptides to differentiate into an elongated and polyploid morphotype.

Bradyrhizobium diazoefficiens USDA110 BclA is also not required for symbiosis with NCR-producing Aeschynomene afraspera

B. diazoefficiens strain USDA110, which is a natural soybean symbiont, can also form functional nodules on A. afraspera29, 32. The strain forms nodules composed of a central zone with fully infected symbiotic cells and a cortex layer surrounding the infected cells. This nodule organization is very similar to the histology of nodules induced by the natural Aeschynomene symbiont Bradyrhizobium strain ORS285 (Fig. S1). Nevertheless, USDA110 is a less efficient nitrogen fixer, supporting lower plant biomass production and nitrogen accumulation than ORS28532 (Fig. S2). The latter strain forms elongated and polyploid bacteroids on A. afraspera and requires the bclA gene for elongated bacteroid formation9. Therefore, we analyzed the bacteroid type formed by strain USDA110 within A. afraspera nodules and the role of the USDA110 bclA gene. Unexpectedly, observations by confocal microscopy showed that USDA110 bacteroids in A. afraspera nodules were not or only very slightly elongated (Fig. 5b), contrary to bacteroids of strain ORS285 which are strongly elongated4, 9. To confirm this unpredicted observation we used flow cytometry analysis of the bacteroid cell size, determined by the forward scatter (a measure for cell size), and the DNA content, measured by DAPI fluorescence. A slight increase in size of the bacteroids compared to the bacteria in culture was measured but this was not accompanied with an increase in the DNA content of the bacteroids (Fig. 5c). Thus the USDA110 bacteroids are much less or hardly differentiated compared to ORS285 bacteroids which have, besides the strong cell enlargement, also a marked increase in DNA content4, 9. The absence of a pronounced differentiation of USDA110 bacteroids is not likely resulting from a defect in NCR gene expression in USDA110-infected nodules since five tested NCR genes were expressed at similar or even higher levels in USDA110-infected nodules compared to ORS285-infected nodules (Fig. S3).

In agreement with the absence of differentiation of the wild type USDA110, the USDA110ΔbclA mutant was not affected in symbiosis with A. afraspera: nodules infected with wild type or mutant were similar, supported plant growth and fixed nitrogen to the same extent (Fig. 5a,d), both types of nodules contained symbiotic cells which were completely infected with bacteroids that seemed not or only slightly elongated (Fig. 5b; Fig. S1) which was confirmed by flow cytometry (Fig. 5c).

BclA is not required for the formation of differentiated bacteroids in Aeschynomene afraspera nodules by the USDA110 DD-CPase1 mutant

Contrary to the wild type USDA110 strain, the UDSA110 DD-CPase1 mutant forms strongly elongated and polyploid bacteroids in the A. afraspera nodules29 (Fig. 5b,c), indicating that by affecting their cell wall strength, the bacteria become sensitive to the NCR differentiation signals produced by the nodule cells. Nevertheless, the nitrogenase activity of plants infected with the UDSA110 DD-CPase1 mutant is strongly reduced, in large part because the mutant induces much less nodules than the wild type29 (Fig. 5d). We created a bclA/DD-CPAse1 double mutant to determine whether the cell wall-determined bacteroid differentiation is depending on the BclA peptide transporter. Unexpectedly, the double mutant exhibited a similar symbiotic phenotype than the DD-CPase1 single mutant (Fig. 5b–d). This result indicates that the differentiation of USDA110, made possible by the inactivation of the DD-CPase1 gene, is independent of BclA.

A possible explanation could be that the DD-CPase1 mutation increases the permeability of cells for peptides, including NCR peptides, rendering the BclA transporter superfluous. To test this possibility, we measured sensitivity of strains against the peptide bleomycin which needs to be internalized to target the bacterial DNA. We found that the DD-CPase1 mutant strain displays a sensitivity to bleomycin similar to the wild type strain (Fig. 5e), indicating that the peptidoglycan remodeling, induced by the mutation, does not interfere with peptide uptake. Similarly, the double mutant is just as much or even slightly more resistant to bleomycin than the bclA mutant (Fig. 5e).

BclA and DD-CPase1 in Bradyrhizobium strain ORS285 act independently in bacteroid differentiation

To further explore the interdependence of BclA and DD-CPase1 in bacteroid differentiation, we created the double mutant also in Bradyrhizobium strain ORS285. This strain forms nodules on A. afraspera as well as on A. indica in which it differentiates into either elongated polyploid or spherical polyploid bacteroids respectively4. A bclA mutation in this strain blocks the differentiation process in both hosts9 (Fig. 6a) while a DD-CPase mutation induces hypertrophied bacteroids29 (Fig. 6a). In a similar way as in USDA110, the ORS285 double mutant was still capable to induce strongly enlarged bacteroids in nodules of both A. afraspera and A. indica (Fig. 6a). In A. indica, the bclA mutation induced death of the bacteria as revealed by the red fluorescence in the live/dead staining procedure of nodule sections (Fig. 6a). Even if in the double mutant, many bacteroids were strongly enlarged (Fig. 6a), others remained undifferentiated. This may be related to bacterial death induced by the bclA mutation in such a way that many bacteria die before having the chance to enlarge. Thus, these results indicate that the bclA and DD-CPase1 mutations have a cumulative effect and that the bclA function is not upstream of the bacterial differentiation provoked by the DD-CPase1 mutation. The cumulative effect of the two mutations is also observed when measuring with the acetylene reduction assay the nitrogenase activity of A. afraspera nodules (Fig. 6b). The bclA mutation has a stronger impact on nitrogen fixation than the DD-CPase1 mutation and the double mutant has the same low nitrogenase activity as the bclA single mutant while on the other hand bacteroids of the double mutant resemble morphologically more the DD-CPase1 bacteroids.

Patterns of cell growth and differentiation in cell layers can influence the quality of mature fruit. For example, pepino fruit with a compact exocarp composed of tightly packed cells are less likely to bruise during postharvest handling than cultivars having large intercellular airspaces. As cell size increases during development, other accompanying characteristics also change, such as cell wall thickness, differentiation of specific cell types (e.g. sclereids) and the formation of cell inclusions such as oil droplets or calcium oxalate crystals (raphides). In feijoa and pear, development of sclereids in the mesocarp provides the characteristic gritty texture. As another example, juiciness of orange depends on prior differentiation of juice sacs in the endocarp.

The extent and distribution of airspaces are particularly important, affecting both fruit texture and physiological properties. For instance, in apple, airspace relative to fruit volume can double during development, while cell wall thickness and relative cell surface area both decline (Figure 11.4). Such changes affect gas exchange and diffusion of solutes through pericarp tissues due to increased tortuosity.

In kiwifruit all tissues of the mature fruit (exocarp, outer and inner pericarp and central core) are already discernable in the ovary before anthesis and pollination. Each layer grows to a different extent and at different rates, so that the relative contribution of each to the total fruit volume varies with time (Figure 11.5). Cell division ceases first in the exocarp and last in the innermost regions of the central core. The outer pericarp is first seen as a homogeneous population of cells but by c. 14 d after pollination two cell types become visible, namely small isodiametric parenchyma cells full of starch grains, and much larger heavily vacuolate ovoid cells in which the frequency of starch grains per unit volume is low.

Fruit anatomy affects our perception of fruit quality. In kiwifruit, hairs are developed as multicellular projections of the skin, giving a characteristic bristly appearance and rough feel in the case of the green flesh ‘Hayward’ or a silky, smooth appearance in the yellow flesh ‘Hort16A’ cultivar. Tough skin relative to soft flesh is another important character imparted by development of primary cell wall thickenings in the hypodermal collenchyma.

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