How to break down cell walls in sugarcane

One way of improving the usefulness of a plant crop might come not from studying where the plant cells grow, but where they die.

There’s a lot of energy inside plants, but it’s not always easy to access it. The cell walls are built from cellulose, which keeps the plant cells rigid. Unfortunately, cellulose is notoriously hard to break down. Some animals can process it, but it’s not simple. A new paper, by Adriana Grandis and colleagues, looks at how sugarcane can break down cellulose to form the aerenchyma.

Spaces in the aerenchyma. Image: Marcos Buckeridge and Débora Leite

Aerenchyma is not stiff. It is a spongy tissue full of spaces and channels for air. Prof. Marcos Buckridge, one of the authors of the study, explained why aerenchyma was so crucial for sugarcane. “Root aerenchyma is thought to be related to an increase in the capacity to keep oxygen flowing inside the root tissue. It thus protects the root against the hypoxia (lack of oxygen) provoked by waterlogging or flooding. We have been searching for sugarcane varieties without aerenchyma, but have not found it yet, at least in the Brazilian varieties that we investigated. Many grasses, including rice, sorghum, and maize form aerenchyma. Maize has been more thoroughly investigated, and in its case, aerenchyma in the roots will only form when it is flooded or waterlogged. Rice, sorghum and sugarcane form root aerenchyma independently of induction by an external signal (i.e., aerenchyma is constitutive).”

One way of getting a better idea of how aerenchyma works would be to alter a plant, so it has less of them. And this is work Prof Buckeridge’s team have been working on. “We have been trying to suppress aerenchyma formation in sugarcane. We tried, for instance, adding substances that inhibit signalling of the hormone ethylene (Tavares et al., 2018), which is well known to induce aerenchyma formation, but could not do it yet. We also tried to increase a repressor of the first step (pectin degradation), the transcription factor scRAV1 (characterised by Tavares et al., 2019), but found that there are mechanisms that seem to “protect” aerenchyma formation, in this case being a micro RNA that target the transcription factor.”

“It is possible that aerenchyma is important also for growth improvement in sugarcane, as it may provide oxygenation of the roots, independent of flooding or waterlogging. It is well known by breeders that deeper and fast-growing roots improve sugarcane production. The aerenchyma might have been blindly selected by breeders, as they searched for higher growth, sugar, and biomass production.”

It’s the way that aerenchyma form that makes them such a useful feature to study. The sugarcane doesn’t grow with these air channels pre-formed. Instead, there’s a process of demolition inside the plant at it grows. Prof. Buckeridge said this might already be familiar to Botany One readers. “The formation of the aerenchyma can be divided into modules (Grandis et al., 2014, Tavares et al., 2015). The phenomenon is described in Leite et al. 2017, and a film of its formation within the root is available for download as supplemental material or directly from Botany One. The first step is signalling. Probably a single cell of the cortex “senses” the balance between ethylene (produced locally) and auxin (coming from the leaves) and enters the second stage which is characterized by two features: cell separation (attack of enzymes to the middle lamella) and cell expansion. At the same time, programmed cell death starts (see also the earlier article at Botany One). As cells die, they produce enzymes synchronically to modify cell wall composition (Grandis et al., 2019), forming a composite (Leite et al., 2017) which becomes gradually recalcitrant to the hydrolases (Grandis et al., 2019) and end up creating channels that supposedly are impermeable to gasses and form a series of interconnected pathways that conduct oxygen through the root.

While examining how aerenchyma forms might seem esoteric, it’s a valuable question. There could be a big pay-off from understanding how it forms. Prof. Buckeridge said: “Our paper could be interesting for those who would like to know more about how can we manipulate plants to produce more bioenergy from plant biomass. To do that, it is essential to get the sugars from substances like cellulose. By breaking it, one produces glucose that can be given to yeast that ferments the sugar and produces ethanol. However, this is very difficult because of the cell walls (the composite lying outside of all plant cells) is much more complicated and have several other polymers besides cellulose. Our paper describes how genes are activated to produce proteins (enzymes) that can break those polymers. This is part of a strategy to make sugarcane plants to behave like a fruit, becoming soft and easy for the industry to obtain the sugars for ethanol production.”

Enhancing this process of converting sugars to ethanol could help reduce carbon dioxide emissions. Prof. Buckeridge said, “Sugarcane is one of the leading bioenergy crops on the planet. Besides being the primary source of sugar (sucrose) used for food purposes, it is also one of the primary sources of ethanol for use as a biofuel. The expansion of sugarcane in Brazil without any effect on food production or on conservation of biomes, including the rain forest, would be enough to displace up to 6% of the gasoline used in the plant and also decrease emissions of CO2 by up to 14% on the basis of 2014 (Jaiswal et al., 2017). To achieve goals like this, we would need to use not only the free sugars (sucrose) already available in sugarcane tissues but would need to obtain the sugars from the cell walls. Thus, one of the critical processes to produce ethanol from the biomass of plants is called hydrolysis. This latter process is necessary because more than 60% of the mass (excepting water) of a plant is cell walls. This is what we call 2nd Generation (2G) Bioethanol.”

“Because I worked all my career (now 38 years) with the endogenous process of cell wall degradation in seeds (Buckeridge et al., 2005), when the issue of bioenergy became prominent around the middle of the 2000 decade, I decided to search for developmental processes in sugarcane where the cell walls were being degraded. At the time, one of my PhD students was working with papaya development. We were observing quite interesting processes in which the cells of the fruit separate (the cement – middle lamella – is degraded by enzymes) and the softness of the mature fruit yields the sweet taste to the consumer. I thought that we could perhaps find developmental processes in sugarcane that would be analogous to what I have studied in seeds and fruits and try to use that to reengineer sugarcane to soften like a fruit. From this time on, I named this project the “papaya cane”. We first looked for that in leaf senescence but found no sign of cell wall modification (Martins et al., 2016). We continued looking until we found the aerenchyma. We were impressed because the whole cortex of the roots collapsed, leaving debris of cell walls. My group was then directed to investigate the aerenchyma of sugarcane regarding the events associated with the cell walls. The description of the process was published in 2017 in Annals of Botany, and now we report the mechanisms of gene expression, protein production, and enzyme activity involved in aerenchyma formation.”

“Along with other published chapters and papers (Grandis et al., 2014, Buckeridge and De Souza, 2014, Tavares et al., 2015, and others), we are getting close to what the papaya cane should be. We have good evidence that an attack to the pectins in the middle lamella (like happens in the fruit) is what starts aerenchyma development and could do the job of softening the biomass of sugarcane. Using a transcription factor discovered by Tavares et al. (2019), which controls the first step of the process, we produced genetically modified plants. These are being analysed to see whether they could be close to what we expect the papaya cane to be.”

While the paper answers some questions of how aerenchyma form, there’s still a lot of work to come Prof. Buckeridge said: “One of the main goals of this line of research in my lab is to understand mechanisms that could be used to control the hydrolysis of cell walls to soften the whole body of the sugarcane plant. We now think that this can be done by triggering the first step of aerenchyma formation, i.e., cell separation. This first step alone may be sufficient to help to decrease the demand for energy for the process of 2G bioethanol production from sugarcane.”

“The second goal is related to the production of more efficient hydrolases. Our work is contributing to developing enzyme cocktails that are specific for hydrolysis of sugarcane cellulose and hemicelluloses so that the crushed biomass would be more easily pretreated and hydrolysed. The idea is that the cell wall hydrolases produced by a plant against its own cell walls might be more efficient in an industrial process. From a collection of approximately 1,200 different cell wall-related enzymes from sugarcane that we found until now (to be published), we used the knowledge acquired with the studies we made on the aerenchyma of sugarcane to select two candidate enzymes. We cloned and characterized these genes (an endopolygalacturonase and an alpha-arabinofuranosidase) and expressed them heterologously in yeast. These enzymes are now being characterised, and we intend to add them to enzyme cocktails used in the industry and test the hypothesis that efficiency would increase with plant enzymes.”

“One of the main challenges we have and are still facing is the lack of a full genome sequence of sugarcane. But this has been developing quite well more recently, and we have now access to genome drafts that are helping to get complete sequences, promoters and much more detail of the enzymes. We already have a list of 29 sugarcane hydrolases that appear to be good candidates to be used as additives to commercial enzyme cocktails. With the full sequences obtained from the genome and the subsequent heterologous expression of such enzymes in microorganisms, enzyme cocktails could be improved significantly. Another route would be the activation of enzyme consortia within the sugarcane tissues to promote endo-hydrolysis so that the industrial process could become independent of pretreatments. To achieve this goal, we would need to “install” an aerenchyma formation system in the other organs of the sugarcane plant. As we gather tools and understand mechanisms, I think this is an exciting challenge of synthetic biology for the near future.”

Further reading

Buckeridge, M. S., Hutcheon, I. S., & Reid, J. S. G. (2005). The Role of Exo-(1→4)-β-galactanase in the Mobilization of Polysaccharides from the Cotyledon Cell Walls of Lupinus angustifolius Following Germination. Annals of Botany, 96(3), 435–444. https://doi.org/10.1093/aob/mci192

Buckeridge, M. S., & de Souza, A. P. (2014). Breaking the “Glycomic Code” of Cell Wall Polysaccharides May Improve Second-Generation Bioenergy Production from Biomass. BioEnergy Research, 7(4), 1065–1073. https://doi.org/10.1007/s12155-014-9460-6

Grandis, A., de Souza, A. P., Tavares, E. Q. P., & Buckeridge, M. S. (2013). Using Natural Plant Cell Wall Degradation Mechanisms to Improve Second Generation Bioethanol. Plants and BioEnergy, 211–230. https://doi.org/10.1007/978-1-4614-9329-7_13

Grandis, A., Leite, D. C. C., Tavares, E. Q. P., Arenque-Musa, B. C., Gaiarsa, J. W., Martins, M. C. M., … Buckeridge, M. S. (2019). Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots. Annals of Botany. https://doi.org/10.1093/aob/mcz099

Jaiswal, D., De Souza, A. P., Larsen, S., LeBauer, D. S., Miguez, F. E., Sparovek, G., … Long, S. P. (2017). Brazilian sugarcane ethanol as an expandable green alternative to crude oil use. Nature Climate Change, 7(11), 788–792. https://doi.org/10.1038/nclimate3410

Leite, D. C. C., Grandis, A., Tavares, E. Q. P., Piovezani, A. R., Pattathil, S., Avci, U., … Buckeridge, M. S. (2017). Cell wall changes during the formation of aerenchyma in sugarcane roots. Annals of Botany, 120(5), 693–708. https://doi.org/10.1093/aob/mcx050

Martins, M. T. B., de Souza, W. R., da Cunha, B. A. D. B., Basso, M. F., de Oliveira, N. G., Vinecky, F., … Molinari, H. B. C. (2016). Characterization of sugarcane (Saccharum spp.) leaf senescence: implications for biofuel production. Biotechnology for Biofuels, 9(1). https://doi.org/10.1186/s13068-016-0568-0

Tavares, E. Q. P., De Souza, A. P., & Buckeridge, M. S. (2015). How endogenous plant cell-wall degradation mechanisms can help achieve higher efficiency in saccharification of biomass. Journal of Experimental Botany, 66(14), 4133–4143. https://doi.org/10.1093/jxb/erv171

Tavares, E. Q. P., Grandis, A., Lembke, C. G., Souza, G. M., Purgatto, E., De Souza, A. P., & Buckeridge, M. S. (2018). Roles of auxin and ethylene in aerenchyma formation in sugarcane roots. Plant Signaling & Behavior, 13(3), e1422464. https://doi.org/10.1080/15592324.2017.1422464

Tavares, E. Q. P., De Souza, A. P., Romim, G. H., Grandis, A., Plasencia, A., Gaiarsa, J. W., … Buckeridge, M. S. (2019). The control of endopolygalacturonase expression by the sugarcane RAV transcription factor during aerenchyma formation. Journal of Experimental Botany, 70(2), 497–506. https://doi.org/10.1093/jxb/ery362