Review artic le

Sugarcane for bioenergy production: an assessment ofyield and regulation of sucrose contentAlessandro J. Waclawovsky1,†,�, Paloma M. Sato1,�, Carolina G. Lembke1, Paul H. Moore2 and Glaucia M.Souza1,*

1Departamento de Bioquımica, Instituto de Quımica, Av. Prof. Lineu Prestes, Sao Paulo, Brazil2Hawaii Agriculture Research Center, Kunia, HI, USA

Received 15 June 2009;

revised 19 November 2009;

accepted 20 November 2009.

*Correspondence (Tel +55 11 30918511;

e-mail glmsouza@iq.usp.br)

†Present address: Alessandro J. Waclawo-vsky,

Universidade Tecnologica Federal do

Parana (UTFPR), Campus Dois Vizinhos,

Estrada para Boa Esperanca, km 04, Dois

Vizinhos, PR, Brazil, 85660-000.�The first two authors contributed equally

to this article.

Keywords: sugarcane, biomass, bio-

energy, genes, yield, expression.

SummaryAn increasing number of plant scientists, including breeders, agronomists, physiolo-

gists and molecular biologists, are working towards the development of new and

improved energy crops. Research is increasingly focused on how to design crops spe-

cifically for bioenergy production and increased biomass generation for biofuel pur-

poses. The most important biofuel to date is bioethanol produced from sugars

(sucrose and starch). Second generation bioethanol is also being targeted for studies

to allow the use of the cell wall (lignocellulose) as a source of carbon. If a crop is to

be used for bioenergy production, the crop should be high yielding, fast growing,

low lignin content and requiring relatively small energy inputs for its growth and

harvest. Obtaining high yields in nonprime agricultural land is a key for energy crop

development to allow sustainability and avoid competition with food production.

Sugarcane is the most efficient bioenergy crop of tropical and subtropical regions,

and biotechnological tools for the improvement of this crop are advancing rapidly.

We focus this review on the studies of sugarcane genes associated with sucrose

content, biomass and cell wall metabolism and the preliminary physiological

characterization of cultivars that contrast for sugar and biomass yield.

Energy crops: trait improvement and yield

There has been a research surge in recent years aimed at

developing alternative sources of energy that can decrease

or replace the use of fossil fuel. The fluctuating prices of

petroleum, its dwindling worldwide stocks and the adverse

environmental effects of fossil fuel usage have collectively

renewed interest in the search for alternative sources of

energy. Wood, livestock manure, microbial biomass, agri-

cultural waste, agricultural by-products and crops are a

few examples of biological materials that can be used to

produce bioenergy (FAO, 2000).

Several crops are being tested for bioethanol produc-

tion. Ethanol is produced through the fermentation of

sugars or starch from sugarcane, maize, wheat, sugar

beet, cassava and others. Most of the ethanol produced in

the world derives from plant juice containing sucrose from

sugarcane in Brazil and starch from corn in the United

States (EIA, 2008). Ethanol can also be produced from

sugars derived from lignocellulosic material by hydrolysis

of the cell wall using enzymes, physical and chemical

treatments (Ragauskas et al., 2006). The efficiency of the

lignocellulose conversion process has not yet proven to be

economical, but second generation ethanol is a highly

desired goal because it could significantly broaden the

choice of feedstocks. It would then be possible to use

noncrop plants or crop biomass fractions, that are not

animal feeds or food for humans, for biofuel generation

(Tollefson, 2008).

Potential energy crops include sugarcane, maize, sugar

beet (Beta vulgaris), grains, elephant grass (Pennisetum

purpureum), switch grass (Panicum virgatum), Miscanthus

(Miscanthus giganteus) and others. For commercial mar-

kets to develop, many of these crops are being evaluated

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd 1

Plant Biotechnology Journal (2010) 8, pp. 1–14 doi: 10.1111/j.1467-7652.2009.00491.x

for production under short growing seasons, periodic

drought, low temperatures and low input of nutrients. In

an ideal situation, if the goal is to produce energy from

the C-bonds of plant lignocellulose breakdown, the crop

should be a high yielding, fast growing, with a cell wall

that is easy to break down and requiring relatively small

energy inputs for its growth and harvest. To achieve sus-

tainability, energy crops should not require extensive use

of prime agricultural lands, and they should have low cost

of energy production from biomass. Basically, the crop

energy output must be more than the fossil fuel energy

equivalent used for its production. At present, the crop

that has most successfully met the energy crop attributes

above is sugarcane.

Sugarcane as an energy crop

The output to input ratio of sugarcane first generation

ethanol production is around 8–10, compared to 1.6 in

maize (Goldemberg, 2008). Currently, sugarcane stalks

crushed and extracted for juice are subsequently burned in

the sugarcane factories for the production of steam and

electrical energy. Potentially, with the industry of cellulosic

ethanol it is expected that the ethanol output might

increase from the current 7500 to 13 000 L ⁄ ha, i.e. an

increase of 40%–50%. Sugarcane second generation eth-

anol has not yet been used commercially, but many sugar-

cane breeding programmes are improving germplasm not

only for sucrose yield but also for biomass yield in antici-

pation of upcoming technologies that may allow for effi-

cient energy production from cellulosic residues.

Sugarcane annual production per hectare (39 t ⁄ ha of dry

stalks and trash; discussed below) compares favourably to

other high-yield bioenergy crops such as Miscanthus

(29.6 t ⁄ ha), switchgrass (10.4 t ⁄ ha) and maize (total grain

plus stover, 17.6 t ⁄ ha) (Heaton et al., 2008).

Sugarcane is an important food and bioenergy source

and a significant component of the economy in many

countries in the tropics and subtropics. Nearly 100

countries produce sugarcane over an area of 22 million

hectares—approximately 0.5% of the total world area

used for agriculture (FAOSTAT, 2008). Sugarcane produces

the world’s greatest crop tonnage (FAOSTAT, 2008),

even though each of the major cereals—rice, wheat and

maize—occupies a several-fold larger fraction of the

world’s arable land.

In Brazil, the sugarcane agribusiness accounts for more

than US$ 20 billion ⁄ year and is one of the main direct and

indirect job generation sectors. The country produces 25%

of the world’s cane sugar and is the largest producer

(31 million tons ⁄ year) and exporter (19.5 million tons ⁄ year)

(UNICA, 2009). It is estimated that 558 million tons of sug-

arcane will be produced in 2008 ⁄ 2009 (Conab, 2007). Half

of the cane produced will be destined for bioethanol to

feed an increasing number of flex-fuel cars. In 2008,

2 254 553 flex-fuel car units were sold while gasoline pow-

ered cars amounted to 639 199 (ANFAVEA, 2008). It has

been calculated that to meet demand (internal and exter-

nal) Brazil needs to significantly increase its ethanol produc-

tion (double in 5–7 years). Bioethanol has been produced in

integrated bioethanol and sugar production units from the

depleted syrup after sugar manufacture (in this case it is

postulated to be the ‘one and half’ generation process). In

this way there is very little competition of food vs. fuel. In

addition, sugar production has been in the uprise as a result

of the increasing international prices An increase in ethanol

production without decline of sugar production should be

achieved by expanding the planted area or increasing yield

on existing sugarcane lands.

Sugarcane yields

Sugarcane yield statistics are reported on an area basis as

the mass of the sugarcane stalks delivered to the processing

mill and the mass of the sugar produced from the harvested

sugarcane material. As commercial yield statistics are aver-

ages of all genotypes, across all environments and produc-

tion systems, they give no indication of yields that might be

achieved if identifiable yield-limiting constraints were ame-

liorated. For example, in a given year there might have been

a prolonged drought or a pest or disease outbreak that

reduced that year’s yield below the long-term average yield.

Therefore, it is informative to compare yields under a variety

of production situations from commercial average (the low-

est level of production), to commercial maximum, to experi-

mental maximum (Rabbinage, 1993).

Commercial average yields are those reached industry

wide under various agronomic yield-limiting constraints of

weeds, pests, diseases and soil nutrient deficiencies for

which loss prevention measures are generally available.

Commercial yields are the average of reported yields of all

varieties over all environments and from a full range of

farming practices from the poorest to the best. Under

poor farming conditions, a relatively small input of herbi-

cides, pesticides or soil amendments can raise the average

commercial yields to commercial maximum yields attain-

able under the prevailing environment. Commercial maxi-

mum yields are obtained under good farming practices

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Alessandro J. Waclawovsky et al.2

including the use of good plant nutrition and irrigation

when necessary. Still, commercial maximum yields can be

constrained by cultivars not selected as optimum for the

prevailing environment. Attempting to reduce all environ-

mental constraints in an experimental system to increase

commercial maximum yields to the yield potential is more

difficult and costly than raising the average commercial

yield to commercial maximum yield. Potential yield is the

yield that is achieved when a cultivar is grown in the envi-

ronment to which it is adapted, with water and nutrients

nonlimiting, and with pests, diseases, weeds and other

stresses effectively controlled (Evans, 1994; Evans and

Fisher, 1999). One can consider maximum experimental

yields as approaching or equivalent to potential yields.

Sugarcane experimental stations frequently communi-

cate to the growers they serve yield data, including com-

mercial average, commercial record yields and

experimental maximum yields, as a way to compare local

production to other sugarcane growing areas of the world

and to monitor their own historical progress in crop

improvement. Irvine, (1983) compared the commercial

average and maximum sugarcane yields and then calcu-

lated the equivalent total dry matter production for six

sugarcane producing countries. The yields of the three

highest sunlight countries (analysed by Irvine (1983) -Aus-

tralia, Colombia and South Africa- reveal extremely high

cane fresh weight yields that averaged 84 t ⁄ (ha yr)

(Table 1). The commercial maximum cane yield for these

three countries averaged 148 t ⁄ (ha yr) and the experi-

mental maximum averaged 212 t ⁄ (ha yr). Breeding pro-

grammes have been successfully increasing yield at around

1% a year. Recently, in Brazil, a commercial maximum

yield of 260 t ⁄ ha in 13 months (Fazenda Agrovale, Bahia)

and an experimental maximum of 299 t ⁄ ha were recorded

(Fazenda Busato, Bom Jesus da Lapa, Bahia, RIDESA, per-

sonal communication) which exceed the average maxima

reported by Irvine (1983). These exceptionally high-yield

figures were obtained under irrigation in an area having

low precipitation and low cloudiness hence higher solar

radiation than in most sugarcane producing areas of

Brazil. While the commercial averages and the commercial

maxima are from large land areas and are reasonably

reliable, the experimental maxima are from individual trials

on smaller land areas and thus may be over estimates of

yield over several hectares.

The mature sugarcane plant consists of three main

parts: stalk, leaf and root system. The stalk is composed

by joint segments made up of a node (where the leaf

attaches the stalk and the bud and root primordial are

found) and an internode. The leaf is divided into sheath

and blade, separated by a blade joint. They are usually

attached alternately to the nodes, thus forming two ranks

on opposite sides depending on variety and growing con-

ditions. There is an average total upper leaf surface of

about 0.5 m2, and the number of green leaves per stalk is

around ten. The root system has two kinds of roots: set

roots which are thin, highly branched and shoot roots that

replace the previous as they develop. Shoot roots are

thick, fleshy and less branched and with a limited life.

There is a tillering periodicity where the new tiller (shoots)

develops its own roots to adapt for the changing environ-

ment (Miller and Gilbert, 2009).

The sugarcane crop can be harvested by hand labour, in

which case the material taken to the mill consists of only

the mature culm with the other plant materials left in the

field, or the crop can be harvested mechanically in which

case the material hauled to the mill includes in addition to

the mature culm, some fraction of attached green leaves,

immature culm and part of the blanket of dead leaves

(referred to as trash). Commercial yields are reported on a

fresh weight basis with an attempt to correct for the

amount of trash taken to the mill so that yield data are

comparable. However, to calculate the amount of biomass

dry matter produced, the water content of the crop and

the proportion of milled cane to trash have to be deter-

mined. The amounts of water and proportion of cane to

trash are a function of cultivar, environment and season

of the year the crop is harvested (Donaldson et al., 2008),

so that calculations of biomass must be based on empiri-

cally determined data that are fairly consistent for 1 year

sugarcane crop worldwide. Irvine (1983) used these aver-

ages to calculate t ⁄ (ha yr) dry biomass of 39 for three

Table 1 Average, maximum and theoretical sugarcane yields

(Australia, Colombia, and South Africa) and total dry matter

production

Type of yield

Cane yield Biomass*

t ⁄ (ha yr) t ⁄ (ha yr) g ⁄ (m2 d)

Commercial Average 84 39 10.7

Commercial maximum 148 69 18.8

Experimental maximum 212 98 27.0

Theoretical maximum 381 177 48.5

*Cane yield was converted to biomass dry matter first by calculating stalk

dry wt (t cane ha)1 yr)1 · 0.30) then adding the proportion of trash dry

wt [0.65 (stalk dry wt)] as calculated from Thompson (1978). Except for

the theoretical maximum, table is partial summary of Table 3 of Irvine

(1983). Supplemental Table 1 lists yield in the countries considered.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Improving sugarcane for bioenergy production 3

country commercial averages, of 69 for commercial maxi-

mum, and 98 for experimental maximum (Table 1).

Sugarcane theoretical yield potential

Experimental maximum yield approximates the crop poten-

tial yield limit, which in the case of sugarcane is approxi-

mately 212 t ⁄ (ha year) fresh weight or 98 t ⁄ (ha year) dry

biomass. However, this level of yield remains lower than

the theoretical yield maxima that have been calculated

from models of physiological processes contributing to

plant growth (Monteith, 1977; Loomis and Amthor, 1999;

Long et al., 2006; Zhu et al., 2008). These models are

based on the principles of yield potential (Yp) and primary

production (Pn) at a given incident solar radiation as devel-

oped by Monteith (1977) and presented by Long et al.,

2006, where:

1. Primary production of biomass (Pn = StÆeiÆec ⁄ k) is the

product of the annual integral of incident solar radia-

tion (St), two efficiencies that describe broad physiolog-

ical and architectural properties of the crop, i.e. the

efficiency of light capture (ei) and the efficiency of con-

version of the captured light (ec), and a constant (k)

representing the energy content of the particular plant

mass produced (MJ ⁄ kg).

2. Yield potential (Yp = gÆPn) is the product of primary pro-

duction (Pn) and the harvest index (g) or the efficiency

for partitioning of biomass into the harvested product.

Mean world distribution of daily irradiance recorded

from 1990 through 2004 (published on line <http://

www.soda-is.com/eng/map/#monde>) shows the annual

mean daily irradiance of approximately 230 W ⁄ m2 =

19.872 MJ ⁄ m2 = 198 720 MJ ⁄ (ha d) in the sugarcane pro-

duction areas of Australia, Colombia and South Africa, the

three high sunlight, high mass production per unit area

countries analysed by Irvine (1983). This level of irradiation

can be used with the concepts of Monteith (1977) to cal-

culate a theoretical yield maximum for sugarcane at similar

high solar radiation locations such as the main sugarcane

growing regions of Brazil. Very little of total solar irradi-

ance is available to the plant for biomass production (Zhu

et al., 2008). More than half of the energy is outside of

the photosynthetically active region, and additional losses

are associated with reflection and transmission of the inci-

dent light. Overall, the low efficiencies of light capture (ei)

and poor conversion of the captured light (ec), results in

only about 0.06 of the total irradiance being stored in the

energy of the chemical bonds of C4 plants. Thus, the the-

oretical irradiant energy stored in biomass of sugarcane is

reduced from the 198 720 MJ ⁄ (ha d) striking the earth to

only six per cent of that or 11 923 MJ ⁄ (ha d) providing

that there is maximum leaf canopy interception of photo-

synthetic active radiation (PAR), which is not the case for

the entire crop year as discussed below.

The energy content of plant mass depends on its com-

position with higher quantities of energy stored in fats

and proteins than in simple carbohydrates. Sugarcane is

mainly composed of carbohydrates (sugar and lignocellu-

lose) that have an energy content of (�15.9 MJ ⁄ kg). Thus

the 11 923 MJ ⁄ (ha d) of irradiant energy potentially stored

by sugarcane in biomass having an energy content of

15.9 MJ ⁄ kg calculates to a maximum theoretical primary

productivity yield of 749.87 kg ⁄ (ha d) = 0.750 t ⁄ (ha d) =

273.70 t ⁄ (ha yr) in high sunlight areas. However, a 1-year

sugarcane crop requires approximately 140 days from

ratooning to develop a canopy for maximum interception

of PAR (Singels et al., 2005), and this period of reduced

PAR capture will decrease the year’s total energy for pro-

ducing plant mass by the quantity not captured over 70 of

the 365 days of the year. Thus, the primary maximum

yield would be reduced by 19.2 per cent or 221.2 t ⁄(ha yr). One must keep in mind that yield potential is less

than total primary productivity by the amount of produc-

tivity that does not end up in the harvested product.

Sugarcane has a high harvest index because a majority of

the plant organs are harvested. However, there is a frac-

tion (�0.2) of plant material that remains in stubble and

roots and trash consisting of dead stalks and leaves.

Sugarcane’s harvest index of 0.8 could reduce the crop

primary productivity of 221.2 t ⁄ (ha yr) to a potential yield

of above ground biomass of 177 t ⁄ (ha yr)or a fresh

weight cane yield of 381 t ⁄ (ha yr) (Table 1). Areas receiv-

ing higher solar energy would have a higher potential yield

under optimum growing conditions with selected

cultivars as may be the case in the high yields reported

from Brazil.

The relationships among the production situations can

be used to identify where R&D resources might give the

greatest return in increasing crop yields. Under poor pro-

duction situations, yields will likely be increased by mini-

mizing the effects of reducing factors such as pests and

diseases, and then satisfying the limiting factors such as

water and nutrients. Under advanced production systems,

those external reducing and limiting factors may already

be addressed so that greatest return might be from

research aimed at altering the genetics of the crop plant

to raise the potential yield. It is this potential that the

remainder of this paper is addressing.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Alessandro J. Waclawovsky et al.4

Breeding for energy cane

The Saccharum genus is a group of crop species particu-

larly challenging for improvement. Cultivars are interspe-

cific aneuploid hybrids. The crossing of large genomes

(with multiple recent duplications that allow chromosome

pairing and recombination) makes each progeny genotype

a unique genome. To improve yield and other traits of

interest for the development of an energy cane, research

must unravel the complexities of the sugarcane genome,

develop statistical genetics for highly polyploid genomes

and identify genes associated with sucrose content,

drought resistance, biomass and cell wall recalcitrance.

The reference domesticated species of sugarcane is

S. officinarum, a group of canes with thick and juicy culms

(Daniels and Roach, 1987) which crossed to wild relatives

producing the natural hybrids (S. sinense and S. barberi).

These naturally occurring hybrids were selected and culti-

vated, and sugar extraction probably began from such

hybrids (Daniels, 1975). At the end of the 19th century,

S. spontaneum, a wild species with little sugar and thin

culms was used in the earliest sugarcane breeding program-

mes in search of disease resistant genes to introgress into

S. officinarum to produce cultivars. The interspecific hybrid-

ization solved many disease problems, increased cane yield

and sucrose content (Roach, 1972). All modern cultivars are

derived from a few intercrossing of these hybrids (Price,

1965; Arceneaux, 1967). World collections of germplasm

exist in Florida and India (Naidu and Sreenivasan, 1987;

Schnell and Griffin, 1991; Schnell et al., 1997) that keep

ancestor genotypes and cultivars, and many private collections

of breeding programmes are also kept and used for crosses.

In Sao Paulo state, the biggest ethanol producer in Brazil,

ethanol yield increase can be achieved ideally through the

use of higher sucrose yielding cultivars rather than expand-

ing the sugarcane growing area because sugarcane culture

already occupies 70% of the agriculture land. In 2008, an

expansion into pasture land has occurred in Sao Paulo State

and the Cerrado, the central savannah-like region in Brazil

(Goldemberg, 2008). Cultivation can potentially further

expand into Cerrado and pasture in the centre-west regions

but the sugarcane crop would likely be subject to pro-

nounced drought stress. Drought tolerant cultivars are

highly sought by growers expanding into the northeast as

well. Brazil is indeed seeing an expansion of cane cultivation

into centre-west and northeast regions despite low precipi-

tation and into the south region despite it being too cold for

optimal sugarcane production. This fact, aligned with the

predicted future shortage of water resources, indicates that

a sustained growth of cane cultivation will depend on the

development of high-yielding, drought- and cold-tolerant

cultivars, and adapted to poor soil conditions. Sugarcane

growers in the United States of America would also benefit

from cold-resistant cultivars adapted to poor or sandy soil,

and breeders are crossing sugarcane and Miscanthus geno-

types in search of high-yielding, stress-tolerant hybrids (Lam

et al., 2009). In addition, S. spontaneum has been used as a

source of stress resistance genes because it is well adapted

to harsh climatic conditions (Roach and Daniels, 1987; Ming

et al., 2006). Improving cold tolerance could give growers

an opportunity to extend harvest into the cold season and

to expand production into marginal lands and temperate

regions. Increased cold ⁄ drought tolerance has been

reported for specific hybrids, especially those with germ-

plasm of S. spontaneum, S. sinense and Miscanthus and

having higher fibre content (Irvine, 1977). Increasingly,

S. spontaneum germplasm is being introgressed into breed-

ing lines of programmes in Louisianna, Brazil, Barbados

and Australia (ISSCT, 2009) aiming for higher fibre content

and the breeding of energy cane. Interspecific crosses

followed by backcrossing to established cultivars will,

depending on the selection process, introgress alleles into

commercial types and lead to improved stress tolerance and

a higher fibre content. Expansion into noncrop areas will

likely encounter marginal soils such as sandy, saline ⁄ sodic

or waterlogged soils or those with mineral stress prob-

lems notably having aluminium and manganese toxicity.

Genes for resistance to these soil stress problems are

available in sugarcane cultivars and Saccharum species

(Nuss, 1987). One can expect that new cultivars will be

available in a decade or so to expand cultivation to new

climatic conditions.

Molecular resources for sugarcaneimprovement

The form or phenotype of the commercial cultivars has

changed considerably since the first ancestor Saccharum

genotypes. Originally it was a grass with thin stalks that

accumulated little sugar but it evolved to have thicker culms

with juicier and sweeter internodes. The form of a plant is

frequently associated to changes in regulatory elements

such as cis-regulatory elements (CREs) and transcription fac-

tors (TFs) (Doebley and Lukens, 1998; Costa et al., 2005), a

fact well illustrated by the molecular events associated to

maize (Doebley and Wang, 1997; White and Doebley,

1998) and rice (Li et al., 2006a) domestication. Most of the

traits considered in the selection process of breeding

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Improving sugarcane for bioenergy production 5

programmes have a quantitative nature and are controlled

by many loci (QTL’s), such as Brix rate (soluble solids mea-

sured during plant development), sucrose content, diameter

and number of stalks, fibre content, resistance to pests and

flowering, precociousness, diseases, etc. Some QTLs associ-

ated to stress tolerance code for TFs that control metabolic

pathways (McMullen et al., 1998). TFs have been associated

with tolerance to many stresses including drought and cold

(Yamaguchi-Shinozaki and Shinozaki, 1994, 2005; Kasuga

et al., 1999; Zhu, 2002) and have been studied in sugarcane

by groups researching putative targets for the biotechnologi-

cal improvement of this crop. Recently, QTL discovery has

been aided by the identification of functional markers. A

sucrose synthase-derived marker was associated with a

putative QTL having a high negative effect on cane yield and

also with a QTL having a positive effect on sucrose content

(Pinto et al., 2009). This approach was made possible by the

availability of Expression Sequence Tag (EST) collections.

Several EST collections have been developed (Carson

and Botha, 2000, 2002; Casu et al., 2001, 2003; Vettore

et al., 2003; Ma et al., 2004; Bower et al., 2005) that

helped with gene discovery in sugarcane. The SUCEST EST

sequencing project represents the largest EST collection

and is represented by 43,141 putative transcripts known

as the sugarcane assembled sequences (SAS) http://sucest-

fun.org (Arruda, 2001; Grivet and Arruda, 2001; Vettore

et al., 2003). cDNA microarrays have been used to deter-

mine transcript distribution among sugarcane tissues and

to identify ubiquitous and tissue-enriched gene expression

(Papini-Terzi et al., 2005). Sugarcane culms have been

extensively analysed at the gene expression level. Gene

expression related to sucrose metabolism has been shown

to decrease during culm maturation and genes related to

cellulose, lignin and cell wall metabolism to be modulated

during internode development (Casu et al., 2001, 2003,

2004, 2007; Watt et al., 2005). An in silico analysis of tis-

sue expression patterns for cell wall-related genes has

been done for 459 SAS of the SUCEST database and

reflected quite well the expected physiological characteris-

tics of the tissues (Lima et al., 2001).

The SUCEST-FUN project (http://sucest-fun.org) identi-

fied many genes altered in elevated Brix (percentage of

solids, in sugarcane corresponds mainly to sucrose) and in

response to drought, including TFs and protein kinases

(PK), in a large number of cultivars and genotypes (Rocha

et al., 2007; Papini-Terzi et al., 2009). This indicates that

sugarcane breeding programmes when selecting for high

sucrose content have inadvertently selected for gene

expression changes of certain regulatory genes. Gene

expression studies may be helpful in the identification of

eQTLs (expression quantitative trait loci).

Sugarcane is highly syntenic with other grasses even

though there have been extensive rearrangements associ-

ated with polyploidy (D’Hont et al., 1994; Grivet et al.,

1994; Dufour et al., 1997; Glaszmann et al., 1997; Gaut

et al., 2000). Sorghum may present the simplest syntenic

relation to sugarcane (Grivet et al., 1994; Dufour et al.,

1997; Glaszmann et al., 1997; Guimaraes et al., 1997;

Ming et al., 1998; Asnaghi et al., 2000) and is most likely

among the grasses the one to significantly contribute to

sugarcane QTL studies (Ming et al., 2002; Jordan et al.,

2004). The recent release of the sorghum and maize gen-

ome sequence data (http://www.phytozome.net/sorghum;

http://www.maizegdb.org/) will certainly help in sugar-

cane map-based isolation of genes and identification

of regulatory networks, CREs and promoters. Recently,

an international consortium has been formed (http://

bioenfapesp.org) to sequence the sugarcane genome that

may accelerate the development of markers for yield and

abiotic stress tolerance. Sugarcane cultivars have a very

complex genome, a veritable challenge for its sequencing.

With around 760–926 Mbp (monoploid genome) (D’Hont

and Glaszmann, 2001) the basic Saccharum genome is

twice the size of rice (389 Mbp), similar to Sorghum

(760 Mbp), and significantly smaller than maize

(2500 Mbp). On average, a sugarcane locus is represented

by ten alleles. The somatic cell genome (2C) of the

modern cultivar R570 (2n = �115) was estimated to be

around 10 000 Mb (D’Hont and Glaszmann, 2005). With

next-generation sequencing technologies, researchers

expect to have a draft of several sugarcane cultivars

defined in a few years that will greatly enable studies on

genome structure, comparative genomics and marker

identification for agronomic traits of interest.

Physiology and regulation of sucroseaccumulation

Under certain conditions, sugarcane partitions carbon into

sucrose that accumulates in the internodes to up to 50%

of its dry weight (0.7 M) (Moore, 1995a) but there is very

little knowledge on the regulation of this process. Sucrose,

the carbon compound fixed by photosynthesis and translo-

cated from the leaves to various sink tissues may be stored

as sucrose or partitioned between respiration, including

both catabolic (glycolysis) and anabolic (gluconeogenesis)

processes, and an insoluble cell wall component consisting

of cellulose and lignin. Meristematic sink tissues, including

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Alessandro J. Waclawovsky et al.6

the shoot and root apical meristems and the stalk interca-

lary meristems, metabolize the incoming sucrose into a

hexose pool that is used for respiration, building the insol-

uble component, and depending on environmental condi-

tions and age of the stalk internodes, can be synthesized

back into sucrose for storage. During culm maturation

there is a redirection of incoming carbon from the insolu-

ble and respiratory components to sucrose storage. The

cycle of degradation and synthesis of sucrose in the culm

parenchyma tissue is regulated through the activity of fruc-

tose-6-phosphate 1-phosphotransferase, which appears to

direct the rate of sucrose accumulation by limiting glyco-

lytic carbon flux (Groenewald and Botha, 2007). Factors

regulating the amount of sucrose stored are yet to be

determined but include cell water relations and properties

of the sucrose molecule such as its solubility and transport

by membranes (Moore and Cosgrove, 1991). Molecules

that might be determining cell water content when

sucrose is accumulating are aquaporins less expressed in

internodes of high Brix plants (Papini-Terzi et al., 2009)

that in Arabidopsis have been shown to be involved in

carbon partitioning (Ma et al., 2004).

It is important to keep in mind that the sucrose content

of sugarcane is a trait that should be kept high while

production of cellulosic ethanol becomes economical. In

designing the ideal energy cane it will be important to

keep sucrose levels high, increase biomass yield and alter

the cell wall for enhanced saccharification. Accomplishing

these three goals through metabolic engineering would

require altering carbon fixation and partitioning in such a

highly controlled manner that would be extremely com-

plex, if it is at all possible. If bagasse hydrolysis for ethanol

production becomes very efficient, it may be worth sacri-

ficing sugar for increased fibre content. Either way one

must understand carbon partitioning control mechanisms

to devise strategies to alter it. Transcription factors and

protein kinases are greatly important in defining signalling

and gene networks by regulating key steps in signal trans-

duction through phosphorylation cascades and promoter

activation ⁄ inactivation. Knowledge of the regulatory net-

works controlling carbon metabolism will be critical to

increase yield without deleterious effects on sucrose

metabolism. Recently, the putative 1647 unique sugarcane

TFs have been re-categorized (http://grassius.org) and cat-

alogued into 47 categories (Gray et al., 2009; Yilmaz

et al., 2009). Likewise, sugarcane PKs were re-annotated

and catalogued into the SUCAST (Sugarcane Signal Trans-

duction) Catalogue (http://sucest-fun.org). The SUCAST db

contains 1031 PKs categorized based on BLAST, Pfam

(Sonnhammer et al., 1998), SMART (Schultz et al., 1998)

and a phylogenetic approach (Rocha et al., 2007).

When gene expression was compared among genotypes

with high and low sucrose content (Papini-Terzi et al.,

2009), several TFs were found associated with this trait.

Among the differentially expressed TFs were two helix-

loop-helix, two Homeobox Knotted1-homeodomain genes,

one MYB and several TFs responsive to auxin, ethylene

and gibberellin totalling over 20 TFs correlated to sucrose

content. Auxin- and ethylene-responsive TFs such as ARFs

and EILs were consistently associated with sucrose content

together with the genes responsible for the biosynthesis

of these hormones indicating a predominant role for them

in sugar content regulation or responses.

An experiment comparing culm maturation in 30 geno-

types grown in the field identified developmentally regu-

lated genes related to hormone signalling, stress response,

sugar transport, lignin biosynthesis, fibre content, PKs,

PPases and TFs (Papini-Terzi et al., 2009). Protein phos-

phorylation appeared to have a dominant role in the pro-

cess of internode development as noted by the large

number of PKs, particularly from the SNF-related ⁄ SnRK

kinase family of proteins which were differentially

expressed between high Brix and low Brix plants or

between mature and immature internodes and are respon-

sive to ABA and drought (Papini-Terzi et al., 2009). In

yeast and higher plants, these kinases are known regula-

tors of carbohydrate metabolism (Woods et al., 1994;

Barker et al., 1996; Douglas et al., 1997; Halford and

Hardie, 1998; Sugden et al., 1999; Rocha et al., 2007). It

is possible to make a direct parallel of a putative regula-

tory role for an SnRK1 and 14-3-3 proteins in sucrose

accumulation because several members of this family of

proteins were differentially regulated in sugarcane and

together they have been shown to phosphorylate and

inhibit a sucrose phosphate synthase (Toroser et al., 1998;

Sugden et al., 1999). SnRK2 and SnRK3 were also identi-

fied as regulated during culm development including two

osmotic stress-activated kinases (OSA-PK) and three CBL-

interacting protein kinases (CIPK). OSA-PKs and CIPKs

mediate drought, saline and cold stress responses (Boud-

socq and Lauriere, 2005) which indicates that drought

responses and sucrose content may indeed be related. A

CIPK14 from Arabidopsis thaliana has been shown to

contain sucrose responsive elements in its promoters (Lee

et al., 2005) and several sugarcane CIPKs were shown to

be responsive to sucrose when sugarcane seedlings were

exposed to it (Papini-Terzi et al., 2009). SnRKs are respon-

sive to ABA and drought (Boudsocq and Lauriere, 2005)

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Improving sugarcane for bioenergy production 7

and ABA, sucrose and drought signalling appear to be cor-

related in sugarcane because a comparison of drought

responses, sucrose responses, high sucrose and low

sucrose plants led to the finding that 30% of the genes

associated with sucrose content are also modulated by

drought including the cane SnRKs and PP2Cs homologous

to ABI1 e ABI2 (Papini-Terzi et al., 2009). ABIs have been

shown to be induced by ABA and to mediate stomatal clo-

sure (Merlot et al., 2001; Tahtiharju and Palva, 2001;

Nambara and Marion-Poll, 2005) which may impinge on

photosynthesis efficiency and yield. This is relevant

because the drought responses are also regulated by ABA

and in sugarcane water stress may be a trigger for sucrose

accumulation (Inman-Bamber et al., 2008).

Stress arising from water deficit, defined as any water

content of a tissue or cell that is below the highest water

content exhibited at the most hydrated state, depends on

the level of the deficit and the rate at which it developed.

When the water deficit develops slowly enough to allow

changes in developmental processes, it has several effects,

the most sensitive of which are a reduction in leaf expan-

sion and the closing of leaf stomata. The photosynthetic

rate of a leaf is typically much more tolerant to mild water

stress than is cell expansion. Photosynthate translocation is

the least sensitive of these responses to water deficit

because it is not reduced until the stress becomes severe.

The differential sensitivity of these developmental pro-

cesses is that the net effect of the onset of drought is to

hasten the accumulation of carbohydrates in the leaves

and in storage sinks of the sugarcane plant (Hartt, 1936).

In sugarcane the accumulation of sucrose in storage

parenchyma is called ripening. The central idea in sugar-

cane ripening by drought is to cause a gradual reduction

of the tissue moisture level to compel the plant through a

series of drought reactions that begin with reducing cell

expansion and the formation of new internodes without

much inhibition of photosynthesis. The outcome of this

reduced consumption of sucrose for metabolic energy and

the formation of new cells is an increased sucrose content

(Gosnell and Lonsdale, 1974). Sugarcane crop managers

commonly use drought and other growth-inhibiting stres-

ses to ripen the crop just prior to harvest.

Gene expression differences in cultivars that

contrast for sucrose and biomass

It is possible that some of the genotypes analysed by Papin-

i-Terzi et al., (2009) also differ in biomass content. A con-

tinued agronomic evaluation is necessary to assess how

gene expression in the selected genotypes is related to

other characteristics, such as cell wall composition, growth

rates, internode size and width, number of internodes and

drought tolerance. With this in mind, we started an evalua-

tion of many parameters in addition to Brix among culti-

vars. Sucrose accumulation dynamics, for instance, varies

among cultivars. Breeding programmes have selected for

early and late accumulators that will reach high Brix at dif-

ferent periods of the season. We illustrate this showing

preliminary data for four cultivars that were compared for

sucrose accumulation through the season in both plant

and ratoon crops, sucrose content along internodes in dif-

ferent developmental stages, internode width, biomass

accumulation and plant height (Figure 1). Cultivars SP91-

1049 (V2) and SP89-1115 (V4) accumulate sucrose early in

the season and can be harvested from March onwards

while SP83-2847 (V1) and SP94-3116 (V3) correspond to

varieties with low Brix accumulation rates to be harvested

late in the season (Figure 1a,b). When Brix measures were

taken along the culm, we observed that these varieties also

differ in the internode developmental pattern. V1 and V3

show a sucrose accumulation delay in the first internodes

and do not reach the same high levels of accumulation

in the mature internodes as observed for V2 and V4

(Figure 1c). In addition, V2 and V4 have thicker culms

(Figure 1d) and produce more culm mass (Figure 1e)

without an apparent change in leaf mass (Figure 1e),

plant height, number of internodes or internode length

(Figure 1f).

To verify whether genes, specially protein kinases, previ-

ously seen to be associated with sucrose content in geno-

types (Papini-Terzi et al., 2009) had altered expression in

commercial cultivars (V1, V2, V3 and V4), gene expression

was determined by quantitative real-time PCR (qRT-PCR).

High Brix cultivars exhibited increased expression of two

SnRK2 (CIPK-8 and CIPK-16) and a SnRK1 (SnRK1-2), con-

firming a role for these PKs in sucrose or biomass accumu-

lation (Figure 2). The data implicate also the regulatory

subunits of the SnRK1 kinases (AKINbk) (Bouly et al.,

1999; Lumbreras et al., 2001) in sucrose content regula-

tion or biomass accumulation.

PK expression differences appear to be correlated to cell

wall biosynthesis and expansion as well because a cellulose

synthase (from the CesA family) and a UDP-glucose dehy-

drogenase were observed to be more expressed in the

high Brix ⁄ high biomass cultivars (Figure 2). UDP-glucose

dehydrogenase is responsible for the double oxidation

(four electrons) of UDP-glucose, producing UDP-glucuronic

acid which is a substrate to pectin and hemicelluloses

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Alessandro J. Waclawovsky et al.8

synthesis. This enzyme has been characterized and

extracted from young internodes of sugarcane stems

(Turner and Botha, 2002). In the SUCEST database there

are 52 known cellulose synthase genes belonging to CesA

and CsI family. In the study conducted by Casu et al.,

(2007) they identified 119 transcripts differentially

expressed during culm development. Some members of

the CesA gene family were found to be coregulated, and

two major patterns of expression were detected: high

expression in the maturing stem or high expression in both

maturing and mature stems showing a relationship with

the primary cell wall synthesis. The members of the CsI

gene family were consistently more abundant in the

young stem. The cellulose synthase shown in our study as

associated with sucrose content and biomass is not one of

the family members described by the Casu and colleagues’

work.

Targeting the cell wall

It is evident that if sugarcane is to be improved for bioener-

gy production, a significant number of cultivars and geno-

types need to be further evaluated at the biochemical and

physiological level. It is possible, for instance, that high Brix

genotypes differ in their saccharification potential (i.e. some

may be more amenable for acid and enzymatic hydrolysis

and cellulosic ethanol production than others). Several

genes with a putative function in cell wall metabolism

(a)

(c) (d)

(f)

(b)

(e)

Figure 1 Comparison of the total soluble solids content (Brix) (a, b and c) and developmental performance (d, e and f) in four sugarcane varieties:

V1 (SP83-2847) and V3 (SP94-3116) correspond to varieties with low Brix accumulation rate and late harvest genotypes while V2 (SP91-1049)

and V4 (SP89-1115) correspond to high Brix accumulation rate and early harvest genotypes. Four different plants for each variety were used in

the soluble solids content and developmental performance measurements. The measures of Brix were done with cane plant (a) and cane ratoon

(b) and all developmental (d, e and f) and Brix (c) analyses were done with six-month-old plants. Brix measurements were done using a portable

refractometer. Plant height was measured from the base of the culm until the end of the longest leaf. Dry mass was obtained by drying all the

leaves or all the culms from each of the four plants separately in an oven until constant weight was achieved. The data for plant height, culm

length, internode number and dry mass were analysed by one-way analysis of variance followed by tukey test at 0.05. Means with the same letter

are not significantly different, and means with different letters are significantly different.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Improving sugarcane for bioenergy production 9

were identified as associated with sucrose content (Papini-

Terzi et al., 2009) such as the expansins. Expansins may act

in the relaxation of the cell wall, possibly by breaking the

bonds between cellulose microfibrils and matrix polysaccha-

rides (McQueen-Mason and Cosgrove, 1994; Cosgrove

et al., 2002) allowing for cell expansion. These observations

are corroborated by the identification of an XTH altered at

the expression level during culm maturation. XTHs can

hydrolyse xyloglucans, major components of plant cell walls

and transglycosylate residues into growing xyloglucan

chains that may be important during tissue expansion for

sucrose accumulation (Farrokhi et al., 2006). It is important

to note though that the structure of the sugarcane cell

wall is currently unknown. A preliminary analysis of the

composition of the fibres found a relatively high proportion

of arabinoxylan with cellulose along with lower amounts of

beta-glucan and pectins (Silva, 2005), but studies on the

sugar linkages and overall architecture of the wall have not

been reported yet. Silencing of lignin biosynthesis genes

has been shown to benefit sugar release for lignocellulosic

biomass fermentation (Chen and Dixon, 2007); thus, it

will be interesting to test whether altered biomass has

been selected for during the breeding process at the

level of cell wall architecture, polymer architecture or

cell wall expansion. At any rate, the alteration of cell

wall biosynthesis genes in association with Brix content

is an interesting indication of a correlation between

these processes. Silencing or over-expression of some of

these genes may lead to altered cell wall or increased

sucrose content.

(a)

(c)

(e) (f)

(b)

(d)

Figure 2 Comparison of the mRNA expression of cell wall metabolism enzymes (a and b) and protein kinases (c, d, e and f) in four sugarcane

varieties (V1, V2, V3 and V4; the same varieties of Figure 2). Gene expression data were obtained from total RNA extracted from internode 1 of

twelve-month-old plants that was used as template for cDNA synthesis. qRT-PCR was done as described in Rocha et al. (2007), with a pool from

four different biological replicates for each variety. Each point represents an average (± standard error) of there technical replicates. The gene

expression ratio was analysed by a log-normal model, and the probability P was calculated in relation to a reference (V1 was the reference for V2

and V3 was the reference for V4). The P ‡ 0.95 denotes significant differences on expression levels of V2 and V4 from V1 and V3 (reference),

respectively. The P value was also higher than 0.95 if the references were inverted (V1 as the reference of V4 and V3 as the reference for V2).

Low Brix = low Brix accumulation varieties; High Brix = high Brix accumulation varieties.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 1–14

Alessandro J. Waclawovsky et al.10

Future prospects

Although a lot is known on sugarcane’s biology and culti-

vation, we are only at the beginning of the detailed bio-

chemical and genetic characterization needed for this crop

(Moore, 1995b). There is an impending need to develop

biotechnology that will allow for a sustained industry of

sugarcane. Solid knowledge on photosynthesis, sucrose

and biomass accumulation processes can only be achieved

by combining multiple experimental approaches coupled

to the computational analysis of large sets of data.

Research is imminently needed in sugarcane to pave the

way for a systems biology approach. Ongoing efforts are

aimed to use EST and whole genome sequencing data,

transcriptome analysis, functional data on genes by the

analysis of transgenics and a thorough characterization of

sugarcane varieties including growth, development, physi-

ology in response to stress and agronomical ⁄ industrial

description to build a biotechnology platform for this crop.

To our knowledge, there are no commercial transgenic

sugarcane cultivars. If we take into account that sugarcane

is capable of increasing sucrose up to 25% more than what

is currently available (Grof and Campbell, 2001), we predict

a great benefit if we can successfully target sucrose metabo-

lism genes for increased accumulation. In parallel, we also

need to keep increasing yields for the generation of an

energy cane. Traditional breeding can be improved consid-

erably if the breeders have biotechnological tools available,

such as genes that could be used as markers in the selection

of genotypes. QTLs have been associated with sucrose con-

tent in sugarcane (Silva and Bressiani, 2005; Pinto et al.,

2009) but not mapped yet to TFs. With similar tools as those

used in Arabidopsis (Davuluri et al., 2003; Molina and

Grotewold, 2005; Palaniswamy et al., 2006) and in mouse

and human (Jin et al., 2006; Li et al., 2006b), we will possi-

bly be able to identify polymorphic promoters and CREs that

will be of great importance in the study of sugarcane regu-

latory networks and in the generation of an energy cane.

Acknowledgements

This work was funded by FAPESP (Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo). G.M.S. is recepient of a

CNPq fellowship. We are indebted to Marcio Barbosa,

Geraldo Verıssimo, Marcelo Menossi, Eugenio Ulian,

William Burniquist, Sabrina Chabregas and Maria Cristina

Falco and Cane Technology Center (CTC) for their valuable

help in discussing experiments and data.

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