Cellular Differentiation In Plants And Other Essays On Friendship

Research in Developmental Biology and Plant Physiology - (1964-1985)

From 1964 to 1974, I worked in the Department of Biochemistry at the University of Cambridge, at first as a graduate student, then as a Research Fellow of Clare College Cambridge and as a Research Fellow of the Royal Society. In 1968, and again in 1971, I did research on tropical plants in Malaysia, based in the Botany Department of the University of Malaya, Kuala Lumpur, and also at the Rubber Research Institute of Malaya. From 1974 to 1985, I worked at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in Hyderabad, India, on the physiology of tropical legume crops, first as Principal Plant Physiologist, and later as Consultant Physiologist.

Hormone Production in Plants

The first problem I investigated in my research career was how the hormone auxin (indole-3-acetic acid) is produced in plants. See Hormone Production in Plants below. When I started research on this subject, nobody knew how it was made, and years of efforts had failed to clarify the situation. As I reflected on the biochemical mechanisms by which auxin can be produced I realised that it could be a non-specific breakdown product of the amino-acid tryptophan and that it was likely to be produced in dying cells, as proteins break down, releasing tryptophan. When I started this research, in the 1960's, little attention was paid to dying cells in plants or animals. Nevertheless programmed cell death - also known as apoptosis - is now a very fashionable topic of research in cell biology.

I showed dying cells could produce auxin as a by-product of their autolysis or self-digestion. See The Production of Auxin by Autolysing Tissues

There was nothing specific about the way it was produced in plants. Auxin was also produced, for example, by autolysing yeast cells, and also by autolysing rat liver. Many of the places in which auxin is known to be produced in plants are places where cells die, for example in germinating seeds, as storage tissues breakdown. Auxin is known to be produced in developing leaves and buds, and its formation is roughly proportionate to the development of veins within the leaf. Veins contain xylem or wood cells, and when wood cells develop, the walls thicken up and the cell contains break down and are dissolved away, so cell death occurs in all young growing tissues. Perhaps the differentiating xylem cells were a source of auxin, released as they died.

I studied this question in stems in which new xylem cells were being formed as a result of cambial activity, and found that these thickening stems did indeed produce auxin. See The Production of Auxin by Tobacco Internode Tissues

I also looked at auxin production in senescent leaves. As they go yellow, the cells breakdown and sure enough I found the auxin levels increased dramatically. See Production of Auxin by Detached Leaves. There was only one supposed site of auxin production in plants in which dying cells were not present, namely the tips of coleoptiles in cereal seedlings. These sheathing structures around the seedling shoot were some of the first organs in which auxin was studied and were of particular importance in the classical literature on auxin production. However, although auxin was present in coleoptile tips, I found there was no persuasive evidence that it was made there, and found that in fact it was probably accumulating there having been carried up from the seed in the sap. See Do Coleoptile Tips Produce Auxin?

The formation of auxin in developing xylem cells in the trunks of trees as they grow would mean that a gradient of auxin would be set up across the cambium, the region of dividing cells that separates the wood from the bark. I directly measured auxin levels in the xylem cambium and young phloem cells, from the inside of the bark and showed that there was indeed such a gradient. This was one of the first chemical gradients to be characterised in either animals or plants of a chemical known to have morphogenetic effects. See Auxin in the Cambium and its Differentiating Derivatives

Since dying cells produce auxin, and since dying cells occur within all higher plants as a result of xylem differentiation this raised an evolutionary question. Had the responsiveness of plants to this cell-breakdown product, acting as a chemical signal of cell death, evolved only after cell death became an integral part of plant grown with the evolution of a vascular system? Or have plants already become sensitive to auxin before the vascular system evolved? In fact it was already known that non-vascular land plants, like mosses and liverworts are sensitive to low concentrations of auxin in the environment. They react by producing root hairs, or rhizoids. If this sensitivity had developed in response to dying cells, it would enable mosses and liverworts to produce rhizoids which increase the surface area for absorption of nutrients, in places where there was decaying organic matter, in other words when nutrients were most likely to be abundant. Is auxin really present in such situations? I examined the humus on which mosses and liverworts were growing both in the tropics and in temperate countries and found that it did in fact contain auxin in quantities sufficient to produce rhizoid formation. This suggested an evolutionary origin for the auxin responses in higher plants. First, plants evolved sensitivity to auxin as a signal of organic decay in the external environment. Later, as cell death became an integral part of plant growth the evolution of the vascular system, this hormonal-response system became internalised and auxin evolved the wide range of signalling roles that it has today. See The Occurrence and Significance of Auxin in the Substrata of Bryophytes

At the end of my time at Cambridge, I published a comprehensive review of research on production of auxin and other hormones in plants, summarising the dying-cell hypothesis. See The Production of Hormones in Higher Plants

Auxin Transport in Plants

In plants auxin is transported from the shoot tips towards the root tips by the polar auxin transport system. I investigated which tissues were most involved in this transport process, and whether the polarity of stems could be reversed: I found it could not be. With my colleague Philip Rubery, I worked out the cellular basis of polar auxin transport. Our hypothesis, the so-called chemiosmotic hypothesis, was subsequently confirmed and is now generally accepted. We predicted the existence of auxin efflux carrier proteins preferentially located at the basal end of cells. These proteins were identified in the twenty-first century, and are now called PIN proteins; they are an important focus for contemporary research on plant development. [link to papers on auxin transport]

Cell Differentiation in Plants

As cells in plants turn into wood cells, called xylem cells, they thicken up their walls, and then the cell contents die and dissolve. The developing xylem cells also dissolve away their end walls, so that the dead, empty cells form tiny tubes through which the sap flow from the roots to the shoots. It seemed to me very likely that as new xylem cells formed and became part of the water-conducting system, the contents of the self-digested cells would be flushed away with the sap, and be carried upwards in it. I analysed the sap from several species of plants to see if it did contain breakdown products and enzymes of the kind likely to be involved in the autolysis, or self-digestion, of the differentiating xylem cells, and found that indeed it did. See Some Constituents of Xylem Sap and their Possible Relationship to Xylem Differentiation

Ageing, Growth And Death

In 1974, I published a paper in Nature on the ageing growth and death of cells in which I put forward a new hypothesis that accounts for many of the facts of cellular senescence and regeneration in plants and in animals. In essence, I proposed that harmful breakdown products build up in cells as they age, and that cells can be regenerated by asymmetric cell division so that one of the cells receives more of these harmful products. Thus one daughter cell will pay the price of mortality while the other is rejuvenated. This kind of asymmetric division takes place in the growing regions of plants, the meristems, and in stem cells in animals. It also occurs in the formation of egg cells in plants and animals. In both cases, the meiotic division of the egg mother cell results in one supremely regenerated cell, the egg cell, and three other cells which soon die. In animals these very mortal sisters of the egg cell are called polar bodies.

Scientific Papers on Plant and Cell Biology

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Hormone Production In Plants

Auxin Transport In Plants

Cell Differentiation

The Ageing and Death of Cells

Crop Physiology

Philosophy of Biology

The Production of Hormones in Higher Plants

Do Coleoptile Tips Produce Auxin?

Auxin in the Cambium and its Differentiating Derivatives

The Occurrence and Significance of Auxin in the Substrata of Bryophytes

The Production of Auxin by Autolysing Tissues

Production of Auxin by Detached Leaves

The Production of Auxin by Tobacco Internode Tissues

Effects of Osmotic Stress on Polar Auxin Transport in Avena Mesocotyl Sections

Carrier-mediated Auxin Transport

The Polarity of Auxin Transport in Inverted Cuttings

Auxin Transport in Secondary Tissues

Effect of pH and Surface Charge on Cell Uptake of Auxin

Polar Auxin Transport in Leaves of Monocotyledons

Rupert's research reports as Rosenheim Research Fellow

Cellulase and Cell Differentiation in Acer pseudoplantanus

A Cellulase in Hevea Latex

Cellulase in Latex and its Possible Significance in Cell Differentiation

Some Constituents of Xylem Sap and their Possible : Relationship to Xylem Differentiation

The Ageing, Growth and Death of Cells

Effect of harvest methods on the second flush yield of : short-duration pigeonpea (Cajanus cajan)

Factors affecting growth and yield of short-duration pigeonpea and its potential

A perennial cropping system from pigeonpea grown in post-rainy season

Effect of seed-grading on the yields of chickpea and pigeonpea

Varietal Differences in Seed Size and Seedling Growth of Pigionpea and Chickpea

Effects of Pod Exposure on the Yield of Chickpeas

Iron Chlorosis in Chickpea Grown on High pH Calcareous Vertisol

Growth and Development of Chickpeas under Progressive Moisture Stress

Comparisons of Earlier- and Later-formed Pods of Chickpeas (Cicer arietinum)

Comparisons of Earlier- and Later-formed Pods of Pigeonpeas (Cajanus cajan)

The Effects of Flower Removal on the Seed Yield of Pigeonpeas (Cajanus cajan)

Growth, development and nutrient uptake in pigeonpeas (Cajanus cajan)

A Hydrodynamical Model of Pod-Set in Pigeonpea (Cajunus Cajan)

Pigeonpea as a Winter Crop in Peninsular India

The Expression and Influence on Yield of the 'Double-Podded' Character in Chickpeas

Some Effects of the Physiological State of Pigeonpeas : on the Incidence of the Wilt Disease

Book – The Anatomy of the Pigeonpea

Three Approaches to Biology

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.

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