A research model for carbon-partitioning in sugarcane

Commercial sugarcane (a hybrid of Saccharum officinarum and S. spontaneum) produces a higher biomass yield than the other major world crops, rice, wheat and maize. However, sugarcane yields worldwide have not improved significantly over the past three decades. Good crop yields depend on ensuring that, at each stage of plant growth, the supply of assimilates from the ‘source’ (leaves) to the ‘sink’ (growing or filling tissues) is optimal. Although sugarcane is one of the most efficient crops in converting solar energy into biomass, commercial yields remain half that of experimental potential.

<>

There are several reasons for inefficient conversion of solar energy into biomass. Of particular interest in sugarcane are reduced photosynthetic rates in the leaves and slowed biomass gain in the culms due to feedback control of the plant’s metabolism by high levels of sucrose and other sugars in the leaves. It is difficult to experimentally manipulate sugar levels without changing light input or damaging leaf and culm tissues. Since in YCS leaf sucrose exceeds normal physiological levels, discovering what causes this could give clues to improving productivity.

<>

Sugarcane turns yellow for various reasons that can now be distinguished from YCS, including herbicide application, nutrition and known diseases. Indications are that the syndrome is a combination of abiotic and biotic factors leading to a physiological disorder. Dr Botha and colleagues have found that YCS is especially associated with altered carbon-partitioning in the leaf. Disruption of the sink–source relationship causes sugars to accumulate in leaves, and when sugar exceeds a critical level it induces senescence. High levels of sucrose in sugarcane leaves are therefore an indicator of compromised crop health.

The source–sink system
How well a plant grows depends on acquiring raw material (carbon fixation and mineral uptake), distributing this through plant organs and coping with environmental stresses. The process known as carbon-partitioning is critical for distributing the energy captured by plants through photosynthesis. In C4 plants like sugarcane, CO₂ is converted into four-carbon sugar compounds. These then enter into chemical reactions that take place in chloroplasts, the plant cell organelles conducting photosynthesis.

Carbon fixed during photosynthesis and converted into sugar in ‘source’ cells is distributed to ‘sink’ cells. Phloem is the tissue that transports the soluble organic compounds (mainly sucrose), made during photosynthesis and known as photosynthates, to wherever they are needed in the plant. The sugars are imported into sink tissues for consumption (providing energy for plant functions) or storage. Some stored sugars provide structural biomass as cellulose, hemicelluloses and lignin.

Sucrose synthesis in source tissue, its translocation and its partitioning between storage, respiration and biosynthesis are systemically coordinated in plants. Not only is sucrose the primary product of photosynthesis and the building block for biomass accumulation but it also serves as a sensitive metabolic switch controlling photosynthesis and carbon-partitioning in the plant. A model for the biochemical process of carbon-partitioning in sugarcane is being developed through research on YCS.

Sugarcane has a unique source–sink system. Stem-sinks store photosynthates as soluble sucrose, which can reach exceptionally high concentrations in commercial sugarcane varieties. Most other plant stems store carbon as insoluble polysaccharides (such as starch or cellulose) with low concentrations of sucrose. In many plants, sucrose is stored (after conversion to insoluble starch) in terminal sink organs such as tubers, grains or fruits, rather than in the stem. Valuable sucrose from sugarcane culms is extracted and purified for use in the food industry or fermented to produce ethanol.

Sucrose serves as a sensitive metabolic switch controlling photosynthesis and carbon-partitioning in sugarcane.

During development, sucrose synthesised in sugarcane leaves is translocated via phloem to internodes (the stem sections that run between leaf-carrying nodes), the storage sink. Sucrose accumulates inside and outside the cell membranes, in the symplast and apoplast respectively. Immature sugarcane tissues partition carbon into protein and fibre, whereas mature culms mainly partition it to sucrose storage. During maturation of commercial sugarcane cultivars, leaf photosynthetic activity decreases, as culm sucrose content increases. Thus, sink regulation of source capacity is taking place.

Sucrose accumulation in sugarcane
In YCS, leaf yellowing occurs in the late stage of sucrose accumulation, senescence is induced and tissue death begins. Normal diurnal changes of sucrose concentrations (low in the morning and high at the end of the day) are absent in YCS affected plants, even before yellowing. So, significant metabolic changes occur well before visual signs. Studies at SRA reveal that these changes include an increase in soluble sugars, a decrease in photosynthetic rate, decreased internal leaf CO₂, decreased conductance through stomata (pores in leaves and stems for gas exchange), uncoupling of the photosynthetic electron transport (PET) chain and altered carbon-partitioning.

<>

The excessive increase in sucrose suggests disruption of phloem transport. Sugar is loaded into the phloem but not exported from the leaf, since the highest levels are found in the midrib and sheath. Expression levels of genes for sucrose transporters and SWEET protein (not previously characterised in sugarcane) are also greatest in these plant parts. The sucrose accumulation could be caused by physical blockage of the phloem (for which there is currently no evidence) or arise because the sink is not using transported sugar fast enough which creates an overflow into the surrounding leaf blade, midrib, dewlap and sheath. Increased sucrose also leads to elevated glucose, fructose and trehalose, sugars that play major roles in metabolic signalling. Furthermore, sucrose synthesis slows down which probably leads to a lowering of available inorganic phosphate (Pi) within chloroplasts. A feedback signalling mechanism involving sucrose in the symplast could result from chronic cellular Pi limitation. Research shows that raised sucrose also alters gene expression of key photosynthetic proteins in leaf cells.

From the model developed so far, YCS symptoms appear to be caused by down-regulation of photosynthesis through Pi limitation leading to chronic inability to export reductant away from the PET chain during cellular sugar accumulation. Down-regulation of genes encoding Photosystem (PS) II and I, cytochrome and CP12 (an essential regulatory protein) results in decreased synthesis of these proteins, which then limits photosynthesis.

Advancing genetic studies of sugarcane
The sugarcane genome has only recently been mapped, owing to sugarcane’s complexity: high polyploidy (more than two-paired sets of chromosomes); aneuploidy (varied numbers of chromosomes); bispecific origin of chromosomes; and structural differences and interspecific chromosome recombinants. A reference genome is now available for researchers. DNA sequencing, development of gene-expression technologies and improved genetic/genomics resources for Saccharum are enabling the regulatory networks of carbon-partitioning to be further elucidated.

<>

Metabolome (low-molecular-weight metabolites produced during metabolism) and transcriptome (messenger RNA molecules expressed from the genes) analyses of the metabolic pathways in the leaves and sink tissues of sugarcane are helping researchers to identify reactions that lead to YCS. Comparing leaf transcriptomes of symptomatic and asymptomatic plants confirms that a complex network of changes in gene expression underpin the observed changes in the metabolome.

Fluorescence and gene expression data from YCS studies indicate that PS II is the sensitive process/component, linked to reduced electron flow producing reduced co-enzyme. The early change in photosynthetic rate is accompanied by changes in the expression of phosphoenolpyruvate carboxylase (PEPC). NADP-malic dehydrogenase expression is more sensitive to the accumulation of sucrose than are NAD-malic dehydrogenase and PEPC. This demonstrates that chloroplast metabolism is down-regulated when sucrose levels rise.

Furthermore, genes in the shikimate and phenylpropanoid metabolic pathways are upregulated in early response to elevated sucrose. This increases caffeoyl-quinic acids and quinate, compounds that provide antioxidants to buffer free radical production in the chloroplast as a result of decreased electron flow to the terminal electron acceptors of PS I. Upregulation of the phenylpropanoid pathway probably shifts carbon-partitioning towards lignins, flavonoids and anthocyanins.

A model for the biochemical process of carbon-partitioning in sugarcane is being developed through research on YCS.

In the early stages of sucrose accumulation, several other changes also occur: significant levels of metabolites indicative of microorganisms that associate with injured tissue, especially where there are significant available carbohydrates; significant increases in caffeoyl/chlorogenic type compounds indicative of wounding and activation of plant defence systems; and increases in amino acids and metabolites indicative of stress metabolism and of disruption of the electron transport system, which is dependent on fast turnover of protein components.

A genomic approach is now being pursued for YCS in sugarcane, using next-generation RNA sequencing to compare and analyse genetic data for affected and unaffected plants from diverse field locations. Genetic explorations of how different tissue samples express different proteins, continues to provide clues to the cause of YCS and to understanding sugarcane metabolism in general.