Accepted Manuscript

Title: High-Biomass C4 Grasses − Filling the Yield Gap

Author: John E. Mullet

PII: S0168-9452(17)30414-4
DOI: http://dx.doi.org/doi:10.1016/j.plantsci.2017.05.003
Reference: PSL 9601

To appear in: Plant Science

Received date: 1-2-2017

Please cite this article as: John E.Mullet, High-Biomass C4 Grasses − Filling the Yield
Gap, Plant Sciencehttp://dx.doi.org/10.1016/j.plantsci.2017.05.003

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Plant Science: Commissioned Review

Title: High-Biomass C4 Grasses - Filling the Yield Gap

John E. Mullet ([email protected])

Department of Biochemistry and Biophysics

Texas A&M University

College Station, Texas 77843

Footnote: In this review, gene names are capitalized and italicized (i.e., EHD1), and protein

products derived from genes are not italicized (i.e., Ehd1).

1
Highlights:

 Agricultural productivity needs to increase significantly by 2050

 High-biomass C4 grasses could help fill the yield gap

 Long vegetative growth duration increases biomass yield and resilience

 Prr37 and Ghd7 repress flowering in long days in C4 grasses

 C4 grass stems accumulate sugars ideal for composition engineering

Abstract: A significant increase in agricultural productivity will be required by 2050 to

meet the needs of an expanding and rapidly developing world population, without

allocating more land and water resources to agriculture, and despite slowing rates of grain

yield improvement. This review examines the proposition that high-biomass C4 grasses

could help fill the yield gap. High-biomass C4 grasses exhibit high yield due to C4

photosynthesis, long growth duration, and efficient capture and utilization of light, water,

and nutrients. These C4 grasses exhibit high levels of drought tolerance during their long

vegetative growth phase ideal for crops grown in water-limited regions of agricultural

production. The stems of some high-biomass C4 grasses can accumulate high levels of non-

structural carbohydrates that could be engineered to enhance biomass yield and utility as

feedstocks for animals and biofuels production. The regulatory pathway that delays

flowering of high-biomass C4 grasses in long days has been elucidated enabling production

and deployment of hybrids. Crop and landscape-scale modeling predict that utilization of

2
high-biomass C4 grass crops on land and in regions where water resources limit grain crop

yield could increase agricultural productivity.

Keywords: High-biomass C4 grasses; food security; biofuels; flowering time; drought

resilience; crop modeling

Table of Contents

1. Challenges and strategies for increasing agricultural productivity

1.1. Critical need and time frame assessment

1.2. Land and water resource constraints on grain crop yield

1.3. Lessons from the history of grain crop improvement

1.4. Emerging opportunities for increasing productivity

2. High-biomass C4 grass development, resilience, and composition

2.1. Long vegetative growth duration increases biomass

2.2. Regulation of flowering time in C4 grasses

2.3. Water/nutrient use efficiency, drought resilience

2.4. Opportunities to enhance high-biomass C4 grass composition

3. High-biomass C4 hybrid production and deployment

3.1. Production of high-biomass C4 grass hybrids

3.2. Deployment of high-biomass C4 grass crops

4. Perspectives

3
1.Challenges and strategies for increasing agricultural productivity

1.1 Critical needs and time frame assessment

World population increased from ~1.8B in 1900 to ~7.1B in 2013 and is projected to

exceed ~9B by 2050. Population growth and rapid development are driving increased

demand for food, animal feed, bioenergy, and other bio-products derived from agriculture

creating an ongoing need for increased agricultural productivity [1]. Higher protein diets

associated with rising standards of living increase the demand for agricultural products

because animals convert plant feedstocks into protein at ratio’s ranging from ~2:1 to ~4:1.

Efforts to increase energy independence, reduce energy costs, and increase environmental

sustainability have led to increased production of biofuels over the past decade, primarily

from grain. In the U.S., these trends have resulted in ~80% of agricultural production [2]

and ~80% of the maize grain crop [3] being used for animal feed and production of biofuels.

Longer term, agriculture may also need to generate some of the hydrocarbons used in the

petrochemical industry when economically recoverable supplies of these feedstocks from

geological sources are depleted.

Over the past century, the need for increased production of food, animal feed, fiber,

and other products derived from crops was met by allocating more land to agricultural

production, increasing inputs, optimizing crop management and improving crop genetics.

Intensification of agricultural production has been accompanied by increased use of water

resources, nitrogen fertilizers, and chemicals that control weeds and pests, intensive crop

management, and the development of crops with biotechnology-enhanced traits such as

4
herbicide tolerance and pest resistance. Despite substantial increases in crop productivity

over the past 75 years, it is estimated that output needs to increase an additional ~60-

110% by 2050 [1]. Given this situation, the slowing rate of grain yield improvement of rice,

wheat, and corn, the major sources of calories for the human population, and the

emergence of yield plateaus [4, 5, 6], is of great concern. Following an overview of

constraints, challenges, and opportunities associated with increasing grain crop yield, this

review will examine the potential for high-biomass C4 grass crops to increase agricultural

productivity in water-limited regions of production.

1.2 Land and water resource constraints on grain crop yield

The most useful land for agriculture is already in production spanning nearly 38% of the

Earth’s land surface area [7]. Allocation of more marginal land to agriculture will yield less,

encroach on wildlife habitats that are sources of valuable biodiversity, and risk disruption

of ecosystem sustainability [7]. Agriculture uses 2600 km3 of water each year mainly for

irrigation, nearly two-thirds of all water used by humans [8]. Irrigation is critical for high

crop productivity because insufficient water supply is one of the most significant

constraints on grain yield especially when water-deficit occurs during the reproductive

phase [9, 10]. Development and deployment of more efficient irrigation systems that

optimize the application, timing, and amount of irrigation to minimize run-off and

evaporation could increase yield by utilizing water supplies more effectively. In many rain-

fed regions of crop production that lack access to supplemental irrigation, limited water

supplies reduce yield to some extent annually, and drought periodically causes severe

disruption of grain production [10]. In the future water supplies for agriculture are likely to

become more restricted due to competition from urban centers and industry and because

5
many historical sources of water used for crop production will be depleted within this

century [8]. In addition, climate variation could increase the frequency of drought in some

highly productive agricultural regions. Taken together, this indicates that a significant

increase in agricultural productivity will be required using land and water resources

currently available for this activity to meet the needs of a growing world population that is

undergoing rapid development.

1.3 Lessons from the history of grain yield improvement

Grain is a highly valued agricultural product because seeds accumulate readily digested

starch, oils, and proteins at high density and store these products in a stable low moisture

state that is ideal for harvesting, transport, storage, and utilization. Efforts to improve grain

yield have been underway since crop domestication began thousands of years ago.

Improvements in grain yield are the result of increases plant biomass yield and/or harvest

index [5]. Retrospective analysis indicates that grain harvest index has been under strong

selection in the major cereal crops, reaching an apparent maximum of ~55-60%. More

recent improvements in grain yield are correlated with increased plant biomass yield with

relatively small changes in harvest index. Increases in grain crop biomass yield have

occurred primarily through selection for higher growth rates, increased planting densities

resulting in improved radiation interception, improved radiation use efficiency,

maintenance of high rates of photosynthesis during grain filling (i.e., functional stay green),

and better adaptation to water-limited environments among other traits [5].

Cereal crop yield has plateaued in some of the world’s most productive agricultural

regions [6] indicating that opportunities for increasing grain crop productivity are

becoming more limited as would be expected following a century of yield improvement.

6
The decreasing rate of grain yield improvement indicates that genetic improvement of

grain crops and associated crop management practices may be reaching limits that will be

difficult to overcome without fundamental changes in plant photochemistry, the

enzymology of CO2 fixation, and/or the linkage between CO2 uptake for photosynthesis and

water loss through transpiration. The sensitivity of grain yield to water-deficit during the

reproductive phase is a particularly important constraint that decreases yield, increases

risk, and impacts where grain crops are grown. Plants adjust the number of seed set based

on plant carbon and nitrogen status early in the reproductive phase ensuring that some

viable seed are produced [11, 12]. As a consequence, grain yield is very sensitive to water-

deficit during the reproductive phase because this constraint has a negative impact on

photosynthesis and carbon status and nitrogen uptake and assimilation [9, 10]. Stay-green

traits that reduce canopy size [13] and mechanisms that limit transpiration under

conditions of high vapor pressure deficit (VPD) [14, 15], shift water use and biomass

accumulation from the vegetative to reproductive phase improving grain yield in water-

limited environments. Traits that increase biomass partitioning to seeds by reducing the

growth of leaves and stems post floral initiation and/or that modify carbon-

status/signaling/sink strength by altering production of trehalose-6-phosphate [16] and

ethylene [17] have also been successful in increasing grain yield in water-limited

environments. However, these traits are unlikely to allow a significant extension of grain

crop growth duration in water-limited environments without associated increases in risk.

This is because an inherent property of grain production is the relative inflexibility of grain

crop development relative to variation in the timing and duration of environmental

constraints that occur each season. To manage risk while maximizing yield, grain crop

7
genotypes with specified flowering times are utilized for each region of production based

on average season length and climate constraints. Once grain crops undergo floral

initiation, the time to flowering, and the interval from anthesis to grain maturity are

relatively constant (i.e., ~30 days each for grain sorghum). The selection of grain crop

genotypes with defined flowering times reduces grain yield in years when adverse

environmental constraints occur during the reproductive phase and minimizes the

potential for greater yield through season extension in years with more optimal growing

conditions. In contrast, high-biomass C4 grasses that can tolerate water-deficit and other

constraints during a long duration vegetative growth phase have the potential for higher

biomass accumulation in years with good growing conditions.

1.4. Emerging opportunities for increasing crop productivity

Crop productivity and utility can be increased by: (i) improving the fundamental genetic

potential of plants to capture light energy, carry out carbon fixation, and to accumulate

biomass under good growing conditions; (ii) enhancing crop resilience to biotic and abiotic

constraints that reduce plant productivity in sub-optimal environments; (iii) improving

crop management, nutrient/water inputs, and weed and pest control; (iv) optimizing the

deployment of crops and other plants within and across regions of agricultural production;

and (v) by increasing sink strength and altering the partitioning and composition of

accumulated plant biomass to enhance yield, improve the logistics of harvesting, transport,

storage, and processing, and increase end-product value. Recent advances in high-

throughput phenotyping, multi-scale modeling, low cost genotyping, and gene-editing

technology have the potential to significantly increase the productivity and value of crops.

A key question is how best to utilize these new technologies for that purpose.

8
 Advanced phenomic platforms generate large geo-referenced datasets comprised of

plant/field images, hyper-spectral or multi-spectral information, and environmental data

that can be used to assess rates of biomass accumulation, leaf area, nitrogen, and water

content, onset of biotic and abiotic constraints, and yield with high temporal and spatial

resolution [18]. Automated phenotyping technology can be used to help screen large

numbers of crop genotypes, identify genetic determinants of crop productivity, and

characterize genotype by environment interactions. This technology, in conjunction with

genotyping-by-sequencing methods, has the potential to synergize efforts to improve crops

through the selection of optimal combinations of naturally occurring gene variants and

trait improvement through gene-editing and pathway/trait engineering.

To take full advantage of these emerging technologies and their potential for

increasing the rate of crop improvement, there is a critical need to identify the crops, plant

functions/traits, and crop management practices that could have the largest impact on crop

yield if further optimized. One approach combines knowledge gained from fundamental

research on photosynthesis and employs modeling to identify canopy architecture traits,

photochemical processes, and biochemical reactions that could be modified to improve

plant productivity [19]. For maximum impact, improvements in photosynthetic efficiency

need to be combined with increases in sink strength and resilience to biotic and abiotic

constraints. Collection of data at field and regional scales combined with crop and

landscape modeling allows the identification of constraints operating at higher levels of

organization and traits/management solutions that could minimize these constraints [20,

21, 22]. This modeling approach indicates there are unexploited opportunities for

improving the productivity of agricultural systems associated with more optimal

9
deployment of current and new types of crops such as high-biomass C4 grasses that could

utilize land, water, and nutrient resources more effectively [22].

Historical improvement in crop genetic potential has occurred principally through

the identification and selection of more optimal combinations of gene variants (alleles)

present in crop germplasm collections. The advent of large-scale crop-breeding programs,

multi-location testing, and intensive crop management enabled steady improvement (~1-

3%/yr) in the yield of the targeted crops over the past 50-70 years. The large-scale

required for successful implementation of this approach reflects the fact that useful genetic

variation in crop germplasm collections is distributed among thousands of accessions each

containing a diverse collection of alleles of >30,000 genes per genome. Genomic

sequencing, low-cost genotyping, and genomic association methods are being used to

accelerate the identification and selection of beneficial alleles in crop-breeding programs.

However, because alleles vary in impact and interaction, effective screening for useful

genetic variation requires the evaluation of very large numbers of allelic combinations in

elite genetic backgrounds in multiple environments, performance testing that requires

many years. The large-scale investment and infrastructure required for this approach to

crop improvement has limited the scope of these activities to a few of the most

economically important crops.

Modern crops were derived from a relatively small number of wild plant species

that accumulated useful end-products although often at low levels prior to domestication.

Genetic improvement of these crops focussed primarily on selection for increased yield and

improved composition of the targeted end-products. Comparison of wild and domesticated

versions of plants that produce grain, cotton fibers, tomatoes, and tubers illustrates the

10
highly flexible nature of plant biomass partitioning among organs and end-products of

varying composition. This approach to crop improvement has been highly successful, but

may have resulted in modern crops with associated yield constraints that are now

challenging to surmount.

Accumulated knowledge and recent advances technology provide opportunities to

accelerate the improvement of a broader spectrum of crops and to develop innovative crop

designs that could potentially increase agricultural productivity. This review specifically

examines the potential for high-biomass C4 grasses to increase agricultural productivity. C4

grasses with the highest capacity for biomass acccumuation were identified as the starting

point for this design and then further selected based on their water/nutrient use efficiency

and resilience to water-deficit since grain crops are particularly sensitive to this constraint.

This is followed by a consideration of how the biomass accumulated by these C4 grasses

could be more optimally partitioned into useful end-products. Biomass in this review refers

to the dry weight of all plant derived structures including roots, stems, leaves and

grain/reproductive tissues. Root biomass and root exudates contribute significantly to soil

carbon and chemistry, an important aspect of sustainable crop production. However,

because root biomass is often not measured in field trials, the term biomass yield refers to

harvestable above ground biomass including stems, leaves and grain when present.

2. High-biomass C4 grass crop development, resilience, and composition

2.1 Long vegetative growth duration increases biomass yield

Some of the highest yielding biomass crops are Sorghum bicolor, Miscanthus x giganteus,

sugarcanes (Saccharum spp.) and Pennisetum genotypes, all C4 grass species characterized

11
by large stems, deep canopies, and long growing seasons [23, 24]. The biomass yield of the

perennial C4 grass Miscanthus x giganteus reached 44-61 Mg/ha at peak biomass

accumulation in some mid-west U.S. locations [25] and record yields for Pennisetum

typhoides (~80 Mg/ha) and Pennisetum purpureum (~88 Mg/ha) occur in regions where

water is plentiful and growing seasons are very long [24]. Similarly, first generation annual

high-biomass sorghum hybrids grown in the south-central U.S. accumulated ~40-50 dry

Mg/ha under optimal growing conditions during a ~180 day growing season [26]. The

high-biomass yield potential of the group of C4 grasses listed above is due in part to the

efficiency of C4 photosynthesis, which also aids crop adaptation to hot and dry

environments, and reduces nitrogen requirements for canopy development due to lower

Rubisco levels per unit leaf area [27]. Biomass yield varies among C4 grasses and types of

C4 grass crops for several reasons including season length, stem sink strength, and shoot to

root partitioning. For example, Miscanthus x giganteus produced more biomass than maize

[28] and switchgrass when these C4 grasses were grown in similar locations in the U.S. due

in part to differences in season length and shoot/root partitioning of biomass. High-

biomass sorghum hybrids with long growing seasons accumulated more than twice as

much shoot biomass (~40-50 Mg/ha) as grain sorghum hybrids in good growing

conditions and ~30% more biomass in rain-fed conditions when these crops were grown

in the same location in south-central U.S. [26, 29]. These sorghum crops differ significantly

in duration of growth, planting density, stature, and biomass partitioning (Figure 1).

Figure 1 could go here

12
 The lower biomass yield of grain sorghum compared to high-biomass sorghum was

due to a shorter growing season and because grain sorghum hybrids have short stems

originally selected to facilitate machine harvesting of grain and to reduce stalk lodging

(Figure 1, left). The increased biomass yield of high-biomass sorghum hybrids was due to

delayed flowering resulting in a significant extension of crop growth duration that

increased total light energy capture, improved radiation interception and use efficiency,

and elevated biomass partitioning (~80%) to 4-5 m stems that exhibit high sink strength

(Figure 1, right) [23, 26]. High-biomass sorghum is planted at a density of ~132,000

plants/ha, higher than optimal for sorghum or maize grain crops (~50-80,000 plants/ha),

contributing to increased efficiency of radiation interception early in the growing season

during canopy development. Approximately 60-75 days after emergence, high-biomass

sorghum has a closed canopy that intercepts nearly all radiation useful for photosynthesis

for the duration of the ~180+ day growing season. Radiation use efficiency (RUE) of grain

sorghum is typically 1.2-1.4 g/MJ. In contrast, following canopy closure and onset of rapid

stem elongation, high-biomass sorghum RUE increased to 2.2-2.4 g/MJ during an extended

vegetative growth phase [26]. Similar high RUE has been reported for tall grain sorghum

genotypes [30] and Miscanthus x giganteus [31], although values are still well below 3.5

g/MJ, the theoretical maximum RUE for C4 grass crops [31]. Modeling indicates that further

improvement in biomass yield could be achieved by reducing light saturation of

photosynthesis at the top of C4 grass canopies [19] through optimization of leaf angle [32]

and by modifying the ratio of light harvesting antennae/PSII as a function of depth in the

canopy.

2.2 Regulation of delayed flowering time in high-biomass C4 grasses

13
Delayed flowering in long days is a critically important trait in high-biomass C4 grasses

because this results in an extended period of vegetative growth high biomass accumulation.

A mechanistic understanding of how flowering is repressed in the high-biomass C4 grass

sorghum has proved useful for optimizing growth duration and for development and

deployment of hybrid crops. The analysis of flowering time regulation in C4 grasses has

been facilitated by seminal research on flowering time regulation in Arabidopsis, C3 grasses,

and other plants that identified a large number of genes and pathways that regulate floral

initiation [33, 34, 35, 36]. Research on flowering time regulation in Arabidopsis, a long-day

plant, led to the identification of the gene FT as the source of florigen, a mobile peptide

regulator of floral initiation [33]. Under inductive conditions, florigen is synthesized in

leaves and moves to the shoot apex where it induces reprogramming of the shoot apical

meristem to produce reproductive structures. Homologs of the FT gene family in C4

grasses such as maize (i.e., CN8) and sorghum (CN8, CN12, CN15) serve the same function

[37, 38, 39].

Most of the high-biomass C4 grasses that are the focus of this review are short-day

plants that exhibit varying degrees of photoperiod sensitivity and delayed flowering in long

days [39, 40, 41]. Among the C4 grasses, sorghum is an ideal genetic system for analysis of

photoperiod regulated flowering due to a wide range of photoperiod sensitivity among

sorghum genotypes, facile genetics, and well developed genomics platform. Sorghum

genotypes with minimal response to photoperiod that flower in ~60-70 days are used for

grain production at higher latitudes that have short growing seasons. However, a large

portion of the ~40,000 accessions in the sorghum world germplasm collection are

photoperiod sensitive and exhibit delayed flowering when grown in long days (>12 h) [40].

14
Genotypes with the highest photoperiod sensitivity originate within 20 degrees of the

equator where small differences in day-length modify flowering times [40]. Numerous

populations derived from sorghum genotypes that differ in photoperiod sensitivity and

flowering times were screened in long-day, short-day and field environments in order to

identify quantitative trait loci, key genes and alleles, and characterize the genetic

architecture of the regulatory pathway (Figure 2, Table 1).

Figure 2, Table 1 could go here

The photoperiod-modulated flowering time regulatory pathway in sorghum

includes three genes that are sources of florigen (CN8, CN12, CN15) and upstream

activators and repressors of these genes that are regulated by photoperiod and output

from the circadian clock (Figure 2, Table 1). Sorghum encodes two genes that activate

CN8/CN12-gene expression and flowering, CONSTANS (CO) and EARLY HEADING DATE 1

(EHD1). CONSTANS, a gene that encodes a CCT-domain protein, was first identified in

Arabidopsis as an activator of FT expression and flowering [33]. In sorghum, CONSTANS is

an activator of EHD1, CN8, and CN12 expression and flowering [42]. The grass specific

floral activator encoded by EHD1 [43] also activates CN8 and CN12 expression and

flowering in sorghum [37, 42]. Delayed flowering in long days is mediated by repressors

encoded by PRR37 and GHD7 [37, 44]. Ghd7 represses the expression of EHD1 [44]

whereas Prr37 represses the activity of CONSTANS post-transcriptionally [42]. In

Arabidopsis, Cdf1 factors repress expression of CONSTANS and FT during the day and the

15
corresponding factors in sorghum are thought to have a similar function although this

needs experimental verification.

Photoperiod regulated flowering in sorghum is modulated by light/day-length and

output from the circadian clock [42] once sorghum exits the juvenile phase. The

mechanism for photoperiodic regulation of floral initiation in sorghum is consistent with

the External Coincidence Model [45], although with some features that are unique to

sorghum and related C4 short-day grasses. In sorghum CONSTANS expression is gated to

the evening phase and in photoperiod-sensitive genotypes, CONSTANS is an activator of

EHD1, CN8 and CN12 expression and flowering in short days [42]. In long days, Prr37

inhibits the ability of CONSTANS to activate EHD1, CN8, and CN12, possibly mediated by a

repressing complex involving CONSTANS [42]. In long days, CONSTANS may also help

repress expression of EHD1 through interaction with Ghd7 as occurs in rice [46]. When

sorghum is grown in long days, PRR37 and GHD7 show peaks of expression in the morning

and in the evening that maintain repression of EHD1, CN8, and CN12 throughout the day

and night. Expression of PRR37 and GHD7 in the evening requires light signaling through

phyB and phyC [42,47]. As days become shorter, evening phase expression of PRR37 and

GHD7 is attenuated leading to de-repression of EHD1 expression, an increase in the floral

promoting activity of CONSTANS, and elevated expression of CN8 and CN12 during the

night that results in floral initiation. CN15 is expressed at lower levels in sorghum but

shows a similar pattern of expression in response to day-length indicating that this gene

may also be regulated through the same pathway [38]. The impact of light on the stability

of floral repressors/activators, an important level of regulation in Arabidopsis, remains to

be investigated in high-biomass C4 grasses.

16
 Sorghum was developed as a grain and forage crop ~5,000 years ago in Africa and

sorghum crops are now used throughout the world. Adaptation of sorghum crops to higher

latitudes was accompanied by a reduction in photoperiod sensitivity and earlier flowering.

Retrospective analysis of PRR37 and GHD7 alleles showed that numerous recessive alleles

of these genes are present in sorghum crops utilized at higher latitudes [48]. Some of the

alleles can be traced to accessions from specific regions of Africa but others originated

following importation of sorghum germplasm into other regions of the world. For example,

the prr37Milo allele first identified in the USA cultivar Early White Milo, is unique and must

have arisen after sorghum was introduced into the U.S. [48]. The USDA Sorghum

Conversion program systematically introgressed recessive alleles of PRR37, GHD7, and one

or more dwarfing genes from BTx406 into tall photoperiod sensitive genotypes from the

sorghum germplasm collection. Analysis of ~500 of the resulting converted lines showed

that ~42Mbp of DNA on chromosome 6 spanning recessive alleles of GHD7, PRR37, and

Dw2 in BTx406 was introgressed into a large portion of the lines converted to photoperiod

insensitivity [49].

While the central genes and overall dynamics of the pathway that regulates

flowering time in sorghum in response to photoperiod have been elucidated, there are

significant gaps in our knowledge of this pathway in comparison to what is known in dicots

and C3 grasses. For example, the transcription factors that mediate light-signaling and

clock-output pathways that activate expression of GHD7 and PRR37 in the morning and

evening have not been identified. Similarly, the molecular mechanisms that enable Ghd7,

Prr37 and Cdf1 repress expression of CN8, CN12 and CN15 in long days are not known.

Moreover, ~50 additional QTL have been identified in sorghum that affect flowering time

17
in long days and/or short days [50]. Sorghum flowering time is known to be inhibited

during the juvenile phase, modified by gibberellin, and temperature, consistent with

complex regulation by multiple developmental and environmental inputs as occurs in other

plants.

2.3 Water/nutrient use efficiency, drought resilience

Water/nutrient use efficiency and drought resilience are essential traits for high-biomass

C4 grass crops that will often be grown without irrigation in regions of production that are

subject to water supply limitations during a portion of the growing season. The

accumulation of biomass during an extended vegetative phase allows utilization of

intermittent rainfall during the season and helps avoid the reproductive phase sensitivity

of grain crops to water-deficit. C4 grasses such as Sorghum bicolor and Pennisetum glaucum

(pearl millet) are well-adapted to hot and dry environments because their germplasm is

derived in part from water-limited regions of Africa and India and crop selection has often

occurred in hot and dry regions of the world. During the vegetative phase, sorghum can

tolerate long periods of water-deficit by slowing growth, closing stomata, increased leaf

rolling/erectness, through osmotic adjustment, accumulation of compatible solutes, and by

reducing transpiration through leaf senescence [51]. Sorghum also produces a thick wax

cuticle on leaf and stem surfaces that minimizes water loss during periods of water-deficit.

Some sorghum genotypes exhibit transpiration traits that limit water use at high vapor

pressure deficit (VPD) when transpiration efficiency is low, increasing water use efficiency,

resilience, and biomass yield [14, 15, 52]. The deep canopies of high-biomass C4 grasses

may contribute to improved water use efficiency because a portion of canopy

18
photosynthesis occurs in shaded leaves at lower VPD. It may eventually be possible to

engineer the induction of CAM photosynthesis in high-biomass C4 grasses in response to

extended periods of water limitation, or timed to coincide with summer drought, to achieve

even higher water use efficiency and drought resilience [53]. Root systems of C4 grasses

such as sorghum can reach depths of 200cm allowing efficient capture of water and

nutrient resources while increasing carbon deposition in soil profiles. In addition, high-

biomass C4 grasses such as Miscanthus x giganteus, sugarcane, and sorghum have higher

nitrogen use efficiency compared to grain crops due to efficient nitrogen recycling within

the canopy during vegetative growth, lower nitrogen requirements for grain production,

and possibly through microbe-assisted N-acquisition from soil profiles [54].

2.4. Opportunities to enhance high-biomass C4 grass composition

The composition of high-biomass C4 grass crops could be enhanced for utilization as

animal feedstocks and for production of bioenergy, biofuels, and specialty products. The

composition of harvested biomass can be altered by changing the partitioning of biomass

between stems, leaves and grain (when present), modifying the relative abundance of

protein, cell walls, non-structural carbohydrates, and membranes/lipids within these

organs, and by altering the composition of cell walls and other sub-cellular constituents.

High-biomass C4 grasses typically accumulate 75-80% of their shoot biomass in stems, 15-

20% in leaves, and the balance in reproductive structures/grain when season length and

environmental conditions permit grain production. High-biomass C4 grasses accumulate

~8 Mg of dry leaf biomass per hectare under good growing conditions [26]. Leaf area

increases during development until canopy closure and the rate of new leaf production

19
equals the rate of senescence of shaded leaves lower in the canopy. High-biomass C4 grass

crops such as sorghum accumulate more leaf area than is required for full light interception,

therefore selection for lower leaf area following canopy closure could potentially improve

yield and increase biomass partitioning to stems. C4 grass leaves contain ~9-10% protein

[55] one reason C4 grass crops are used to produce forage for ruminant animals. Forage

biomass digestibility could be increased by development of high-biomass C4 grass crops

with bmr (brown midrib) traits that alter lignin level/chemistry in cell walls, and/or using

pretreatments that reduce the recalcitrance of biomass [56, 57].

Figure 3 could go here

Stems of high-biomass C4 grasses are an ideal target for composition engineering

because most of the harvested biomass accumulates in these organs, stems function as

temporary storage organs that can accumulate large amounts of non-structural

carbohydrates, and they are metabolically active throughout crop development. The stems

of high-biomass C4 grasses such as sorghum and sugarcane reach 4-5 m in length, 40 mm in

diameter, and can account for ~80% of shoot biomass by harvest [26, 59]. Stems of C4

grasses are comprised of nodes and internodes of varying length and diameter (Figure 3).

Phytomers comprised of nodes, internodes, and subtending leaf/leaf sheaths are produced

at regular intervals by the vegetative shoot apical meristem. Stem growth accelerates once

high-biomass C4 grasses exit the juvenile phase and internodes begin elongating. Stems of

C3 grasses typically elongate in response to floral initiation and mutations in the GA

pathway reduce internode elongation and stem length. In contrast, while floral initiation

20
can stimulate stem elongation in C4 grasses, stems of high-biomass C4 grasses also elongate

during the vegetative phase. The short stature of grain sorghum is due to various

combinations of mutations in Dw1, Dw2, and Dw3 that primarily reduce the length of stem

internodes. Mutations in Dw3 (Br2 in maize), a gene that encodes an ABCB1 auxin

transporter, reduces internode lengths, but also stem sink strength, radiation use efficiency,

and grain yield [30, 59]. Dw1 encodes a plasma membrane protein that regulates internode

cell proliferation through the brassinosteroid-signaling pathway [60, 61]. High-biomass

sorghum hybrids utilize active versions of these genes resulting in longer internodes that

accumulate more biomass compared to grain hybrids.

Plant stems act as temporary storage organs when production of photosynthate

exceeds utilization for growth, respiration, and grain-filling. The accumulation of non-

structural carbohydrates in grass stems contributes to improved grain production and

sustains grain development under adverse conditions [62]. Because deposition of sugars in

stems is usually of short duration, most plants accumulate stem sugars that are easily

remobilized such as monosaccharides, sucrose, and fructans. The ratio of non-structural

carbohydrate to lignocellulosic biomass in sorghum stems varies from ~0.5 during rapid

vegetative growth when cell walls are being synthesized, to ~2.8 when production of new

phytomers stops following floral initiation and stems accumulate large amounts of sucrose

in their vacuoles [63]. Lower expression of a vacuolar invertase and decreased utilization

of photosynthate for stem/leaf growth and cell wall biosynthesis is associated with

accumulation of sucrose in stems [63, 64].

Sugarcane, the most economically important high-biomass C4 grass crop, was grown

on 26 M ha in 2012 and produced 1.83B Mg of high moisture stem biomass. Among the

21
high-biomass C4 grasses, sugarcane has been under selection the longest for high stem

sucrose concentration and yield. Commercial biomass yields of sugarcane in some

locations are approaching theoretical limits (~98 Mg dry weight/ha) [62] and the

concentration of sucrose in sugarcane stems has also reached an apparent maximum of 0.5-

0.7M, accounting for up to ~50% of stem dry weight [62]. Increases in sugarcane yield

over the past several decades have resulted in the selection of late flowering genotypes

with increased stem biomass and minimal amounts of grain. A high level of sucrose also

accumulates in stems of sweet sorghum following floral initiation that can result in sink

limitation [63, 64]. These high-biomass C4 grass crops could be engineered to accumulate

high density polymers such as oils, starch, or mixed-linkage glucans (MLG) in stems,

potentially increasing plant biomass yield by reducing futile cycling [65] and minimizing

feedback inhibition of photosynthesis that can occur in sink-limited plants [66]. Stem

starch/MLG has higher density and may be more stable than sucrose post-harvest

facilitating biomass harvest, transport, storage, and utilization. Sorghum stems have been

reported to accumulate up to ~10% starch [63], therefore genotypes with higher stem

starch content could be selected or engineered by changing pathway regulation, providing

an enhanced source of animal feed and/or feedstock for production of biofuels.

3. High-biomass C4 hybrid development and deployment

3.1 Production of high-biomass C4 grass hybrids

Production of high-biomass C4 grass hybrids is a high priority because heterosis enhances

yield. Hybrid seed crops also provide an annual opportunity to adjust land use, the types

of crops grown, and to rapidly deploy improved genotypes across production systems.

22
However, production of hybrid seed from tall late flowering plants is not feasible on a

commercial scale, one reason why perennial high-biomass C4 grasses such as sugarcane are

propagated from vegetative cuttings. This limitation has been overcome in sorghum using

seed parents and pollinators for production of hybrid seed that are short and photoperiod

insensitive due to recessive genes at different loci in the flowering time/stem elongation

pathways, such that complementation occurs in the derived hybrids, causing hybrids to be

tall and late flowering due to high photoperiod sensitivity [67]. This breeding system

allows production of hybrid seed in locations optimal for seed production with subsequent

distribution of hybrid seed to regions where high-biomass C4 hybrid grass crops are

produced [67]. The architecture of the sorghum flowering time regulatory system is ideal

for designing hybrid crops with a wide range of growth duration and specified flowering

times. This flexibility is a consequence sorghum having two activators of the genes

encoding florigen and repressors of these activators that act in an additive fashion to delay

floral initiation in long days (Figure 2). The existence of a naturally occurring allelic series

of the central genes in the photoperiod-responsive regulatory pathway, and in modifiers of

the pathway, provides the genetic diversity needed to design hybrid crops that delay

flowering for up to ~180 days, or as required for each region of crop production.

3.2 Deployment of high-biomass C4 grass crops

Long duration high-biomass perennial and annual C4 grass crops could be deployed in a

complementary fashion with current short season grain crops to increase overall

agricultural productivity and yield stability [22]. Grain crops are already well suited for

shorter growing seasons at higher latitudes and for environments where stored soil

moisture can only support a short duration crop. However, while maize is optimally

23
designed for water sufficient irrigated and rain-fed regions, recent high resolution yield

mapping studies found that maize is not profitable on some land within these production

regions due to shallow soils, sloped land, and other factors [68]. Landscape modeling

indicates that this type of land could be more productive if used to produce high-biomass

C4 grass crops [22], assuming the infrastructure for efficient low-cost processing of the

derived biomass for its targeted end uses was available.

High-biomass C4 grass crops will deliver maximum benefits in regions of agricultural

production with long growing seasons where rainfall is intermittent throughout the season

or where summer drought occurs on a regular basis, but water is available in the spring

and fall. High-biomass sorghum hybrids are well suited for water-limited regions of

production for several reasons; (i) these crops exhibit good water use efficiency and

drought resilience during vegetative growth, (ii) long vegetative growth duration provides

the crop an opportunity to harvest intermittent rainfall for biomass yield production

throughout the growing season, and (iii) a crop designed for biomass harvest has a lower

risk of failure compared to grain crops. In sub-Saharan Africa, photoperiod-sensitive late

flowering sorghum genotypes may have good utility because this type of crop accumulates

stem and leaf biomass for forage and other uses through the end of the summer when

vegetative plants are drought resilient, and produces grain following summer drought. The

development of high-biomass C4 grass hybrid crops using germplasm adapted to these

regions of Africa could potentially increase the supply of feedstocks for animals and

provide supplemental grain yield where food is in limited supply.

4. Perspectives

24
Predictions that agriculture will not be able to meet the needs of an increasing world

population have been articulated at regular intervals over the past 200 years and

intermittent famines, food shortages, and malnutrition have validated these concerns.

However, technological improvements such as the development of hybrids, innovations

associated with the Green Revolution [69], availability of low-cost fertilizer, and industrial

scale plant-breeding programs have allowed crop yields to increase at a rate sufficient to

meet food and feed requirements, although malnutrition is still widespread, especially in

regions of the world where population is increasing the fastest. While large increases in

crop yield represents a major achievement of the 20th century, recent quantitative

assessments show that a significant agricultural production deficit will occur by 2050

unless new technology, innovative approaches, and crops with increased yield are

deployed in a relatively short time frame. The development of drought resilient high-

biomass C4 grass crops with enhanced yield and optimized composition could help meet

projected demand for animal feed, biofuels, and other non-grain agricultural products

helping to fill the yield gap.

Acknowledgements: The author would like to thank colleagues who provided helpful

feedback on the concepts and information presented in this review. Research in the

author’s laboratory is supported by Texas A&M University, Texas A&M AgriLife Research,

the Perry Adkisson Chair, and funding from federal, state and industry sources.

Disclaimer: The author apologizes for not citing numerous seminal publications in the

fields of study covered in this review due to a restriction on the number of citations.

25
Declaration of interest: The author declares no conflicts of interest.

References

[1] D. Tilman, C. Balzer, B.L. Befort, Global food demand and the sustainable intensification

of agriculture, Proc. Natl. Acad. Sci. 108 (2011) 20260-20264.

[2] B.E. Dale, B.D. Bals, S. Kim, P. Eranki, Biofuels done right: land efficient animal feeds

enable large environmental and energy benefits, Environ. Sci. Technol. 44 (2010) 8385-

8389.

[3] P. Ranum, J.P. Pena-Rosas, M.N. Garcia-Casal, Global maize production, utilization, and

consumption, Ann. N. Y. Acad. Sci. 1312 (2014) 105-112.

[4] D.K. Ray, N.D. Mueller, P.C. West, J.A. Foley, Yield trends are insufficient to double global

crop production by 2050, PLOS One 8 (2013) 1-8 (e66428).

[5] A.J. Hall, R.A. Richards, Prognosis for genetic improvement of yield potential and water-

limited yield of major grain crops, Field Crops Res. 143 (2013) 18-33.

[6] P. Grassini, K.M. Eskridge, K.G. Cassman, Distinguishing between yield advances and

yield plateaus in historical crop production trends, Nat. Commun. 4 (2013) 2918.

[7] J.A. Foley et al. Solutions for a cultivated planet, Nature 478 (2011) 337-342.

[8] K. Strzepek, B. Boehlert, Competition for water for the food system, Phil. Trans. Roy. Soc.

B: Biol. Sci. 365 (2010) 2927-2940.

[9] J.S. Boyer, M.E. Westgate, Grain yields with limited water, J. Expt. Bot. 55 (2004) 2385-

2394.

26
[10] J.S. Boyer et al., The U.S. drought of 2012 in perspective: A call to action, Global Food

Security 2 (2013) 139-143.

[11] Y.L. Ruan, Y. Jin, Y.J. Yang, G.J. Li, J.S. Boyer, Sugar input, metabolism, and signaling

mediated by invertase: roles in development, yield potential, and response to drought and

heat, Mol. Plant 3 (2010) 942-955.

[12] T.R. Sinclair, P.D. Jamieson, Grain number, wheat yield, and bottling beer: An analysis,

Field Crops Res. 98 (2006) 60-67.

[13] A.K. Borrell et al., Stay-green alleles individually enhance grain yield in sorghum under

drought by modifying canopy development and water uptake patterns, New Phytol. 203

(2014) 817-830.

[14] T.R. Sinclair, G.L. Hammer, E.J. van Oosterom, Potential yield and water-use efficiency

benefits in sorghum from limited maximum transpiration rate, Functional Plant Biol. 32

(2005) 945-952.

[15] M. Gholipoor, P.V.V. Prasad, R.N. Mutava, T.R. Sinclair, Genetic variability of

transpiration response to vapor pressure deficit among sorghum genotypes, Field Crops

Res. 119 (2010) 85-90.

[16] M.L. Nuccio et al., Expression of trehalose-6-phosphate phosphatase in maize ears

improves yield in well-watered and drought conditions, Nat. Biotech. 33 (2015) 862-869.

[17] J.E. Habben et al., Transgenic alteration of ethylene biosynthesis increases grain yield

in maize under field drought-stress conditions, Plant Biotech. J. 12 (2014) 685-693.

[18] J.W. White et al., Field-based phenomics for plant genetics research, Field Crops Res.

133 (2012) 101-112.

27
[19] S.P. Long, A. Marshall-Colon, X-G. Zhu, Meeting the global food demand of the future by

engineering crop photosynthesis and yield potential, Cell 161 (2015) 56-66.

[20] C.D. Messina, D. Podlich, Z. Dong, M. Samples, M. Cooper, Yield-trait performance

landscapes: from theory to application in breeding maize for drought tolerance, J. Expt. Bot.

62 (2011) 855-868.

[21] A. Wu, Y. Song, E.J. van Oosterom, G.L. Hammer, Connecting biochemical

photosynthesis models with crop models to support crop improvement, Frontiers Plant Sci.

7 (2016) 1518.

[22] P.L. Eranki, D.H. Manowitz, B.D. Bals, R.C. Izaurralde, S. Kim, B.E. Dale, The watershed-

scale optimized and rearranged landscape design (WORLD) model and local biomass

processing depots for sustainable biofuel production: Integrated life cycle assessments,

Biofuels, Bioproducts and Biorefining 7 (2013) 537-550.

[23] J. Mullet et al., Energy Sorghum - a genetic model for the design of C4 grass bioenergy

crops, J. Exp. Bot. 65 (2014) 3479-3489.

[24] C. Somerville, H. Youngs, C. Taylor, S.C. Davis, S.P. Long, Feedstocks for lignocellulosic

biofuels, Science 329 (2010) 790-792.

[25] E.A. Heaton, F.G. Dohleman, S.P. Long, Meeting US biofuel goals with less land: the

potential of Miscanthus, Global Change Biology 14 (2008) 2000-2014.

[26] S.N. Olson et al., High-biomass yield energy sorghum: developing a genetic model for

C4 grass bioenergy crops, Biofuels, Bioproducts and Biorefining 6 (2012) 640-655.

[27] C.S. Byrt, C.P. Grof, R.T. Furbank, C4 plants as biofuel feedstocks: optimising biomass

production and feedstock quality from a lignocellulosic perspective, J. Integr. Plant Biol. 53

(2011) 120-135.

28
[28] F.G. Dohleman, S.P. Long, More productive than maize in the midwest: How does

Miscanthus do it?, Plant Physiol. 150 (2009) 2104-2115.

[29] S.K. Truong, R.F. McCormick, J.E. Mullet, Bioenergy sorghum crop model predicts VPD-

limited transpiration traits enhance biomass yield in water-limited environments, Front.

Plant Sci. 8 (2017) 335-351.

[30] B. George-Jaeggli, D.R. Jordan, E.J. van Oosterom, I.J. Broad, G.L. Hammer, Sorghum

dwarfing genes can affect radiation capture and radiation use efficiency, Field Crops Res.

149 (2013) 283-290.

[31] C.V. Beale, S.P. Long, Can perennial C4 grasses attain high efficiencies of radiant energy

conversion in cool climates?, Plant Cell & Environ. 18 (1995) 641-650.

[32] S.K. Truong, R.F. McCormick, W.L. Rooney, J.E. Mullet, Harnessing genetic variation in

leaf angle to increase productivity of Sorghum bicolor, Genetics 201 (2015) 1229-1238.

[33] T. Imaizumi, S.A. Kay, Photoperiodic control of flowering: not only by coincidence,

Trends Plant Sci. 11 (2006) 550-558.

[34] Y.H. Song, J.S. Shim, H.A. Kinmonth-Schultz, T. Imaizumi, Photoperiodic flowering: time

measurement mechanisms in leaves, Annu. Rev. Plant Biol. 66 (2015) 441-464.

[35] C. Li, H. Lin, J. Dubcovsky, Factorial combinations of protein interactions generate a

multiplicity of florigen activation complexes in wheat and barley, Plant J, 84 (2015) 70-82.

[36] H. Tsuji, K.-i. Taoka, K. Shimamoto, Florigen in rice: complex gene network for florigen

transcription, florigen activation complex, and multiple functions, Curr. Opin. Plant Biol. 16

(2013) 228-235.

29
[37] R.L. Murphy et al., Coincident light and clock regulation of pseudoresponse regulator

protein 37 (PRR37) controls photoperiodic flowering in sorghum, Proc. Natl. Acad. Sci. USA

108 (2011) 16469-16474.

[38] T.W. Wolabu et al., Three FLOWERING LOCUS T-like genes function as potential

florigens and mediate photoperiod response in sorghum, New Phytol. 210 (2016) 946-959.

[39] Z. Dong et al., A gene regulatory network model for floral transition of the shoot apex

in maize and its dynamic modeling, PLoS One 7 (2012) e43450.[40] P.Q. Craufurd et al.,

Adaptation of sorghum: characterisation of genotypic flowering responses to temperature

and photoperiod, Theor. Appl. Genet. 99 (1999) 900-911.

[41] E. Jensen et al., Flowering induction in the bioenergy grass Miscanthus sacchariflorus is

a quantitative short-day response, whilst delayed flowering under long days increases

biomass accumulation, J. Exp. Bot. 64 (2013) 541-552.

[42] S. Yang, B.D. Weers, D.T. Morishige, J.E. Mullet, CONSTANS is a photoperiod regulated

activator of flowering in sorghum, BMC Plant Biol. 14 (2014) 148.

[43] K. Doi et al., Ehd1, a B-type response regulator in rice, confers short-day promotion of

flowering and controls FT-like gene expression independently of Hd1, Genes Dev. 18

(2004) 926-936.

[44] R.L. Murphy et al., Ghd7 (Ma6) Represses Sorghum Flowering in Long Days: Ghd7

alleles enhance biomass accumulation and grain production, The Plant Genome 7 (2014).

[45] C.S. Pittendrigh, D.H. Minis, The entrainment of circadian oscillations by light and their

role as photoperiodic clocks, The American Naturalist 98 (1964) 261-294.

30
[46] Y. Nemoto, Y. Nonoue, M. Yano, T. Izawa, Hd1, a CONSTANS ortholog in rice, functions

as an Ehd1 repressor through interaction with monocot-specific CCT-domain protein Ghd7,

Plant J. 86 (2016) 221-233.

[47] S. Yang et al., Sorghum phytochrome B inhibits flowering in long days by activating

expression of SbPRR37 and SbGHD7, repressors of SbEHD1, SbCN8 and SbCN12, PLoS One

9 (2014) e105352.[48] R.R. Klein et al., Allelic variants in the PRR37 gene and the human-

mediated dispersal and diversification of sorghum, Theor. Appl. Genet. 128 (2015) 1669-

1683.

[49] C.S. Thurber, J.M. Ma, R.H. Higgins, P.J. Brown, Retrospective genomic analysis of

sorghum adaptation to temperate-zone grain production, Genome Biol. 14 (2013) R68.

[50] E.S. Mace, C.H. Hunt, D.R. Jordan, Supermodels: sorghum and maize provide mutual

insight into the genetics of flowering time, Theor. Appl. Genet. 126 (2013) 1377-1395.

[51] A.K. Borrell, D.R. Jordan, J. Mullet, B. Henzell, G.L. Hammer, Drought Adaptation in

Sorghum, in: J.-M. Ribaut (Ed.) Drought Adaptation in Cereals, Food Products Press,

Binghamton, NY, 2006, pp. 335-399.

[52] S.K. Truong, R.F. McCormick, J.E. Mullet, Bioenergy sorghum crop model predicts VPD-

limited transpiration traits enhance biomass yield in water-limited environments, Front.

Plant Sci. 8 (2017) 335-351.

[53] A.M. Borland, H. Griffiths, J. Hartwell, J.A. Smith, Exploiting the potential of plants with

crassulacean acid metabolism for bioenergy production on marginal lands, J. Exp. Bot. 60

(2009) 2879-2896.

[54] S.N. Olson et al., Energy sorghum hybrids: Functional dynamics of high nitrogen use

efficiency, Biomass and Bioenergy 56 (2013) 307-316.

31
[55] B.E. Dale, M.S. Allen, M. Laser, L.R. Lynd, Protein feeds coproduction in biomass

conversion to fuels and chemicals, Biofuels, Bioproducts and Biorefining 3 (2009) 219-230.

[56] J. Gressel, A. Zilberstein, Let them eat (GM) straw, Trends Biotechnol. 21 (2003) 525-

530.

[57] M.C. McCann, N.C. Carpita, Biomass recalcitrance: a multi-scale, multi-factor, and

conversion-specific property, J. Exp. Bot. 66 (2015) 4109-4118.

[58] A.J. Waclawovsky, P.M. Sato, C.G. Lembke, P.H. Moore, G.M. Souza, Sugarcane for

bioenergy production: an assessment of yield and regulation of sucrose content, Plant

Biotechnol. J. 8 (2010) 263-276.

[59] B. George-Jaeggli, D.R. Jordan, E.J. van Oosterom, G.L. Hammer, Decrease in sorghum

grain yield due to the dw3 dwarfing gene is caused by reduction in shoot biomass, Field

Crops Res. 124 (2011) 231-239.

[60] J. Hilley, S.K. Truong, S.N. Olson, D. Morishige, J.E. Mullet, Identification of Dw1, a

regulator of sorghum stem internode length, PLoS ONE 11 (2016) e0151271.

[61] M. Yamaguchi et al., Sorghum Dw1, an agronomically important gene for lodging

resistance, encodes a novel protein involved in cell proliferation, Scientific Rep. 6 (2016)

doi: 1038/srep28366.

[62] T.L. Slewinski, Non-structural carbohydrate partitioning in grass stems: a target to

increase yield stability, stress tolerance, and biofuel production, J. Exp. Bot. 63 (2012)

4647-4670.

[63] B. McKinley, W. Rooney, C. Wilkerson, J. Mullet, Dynamics of biomass partitioning,

stem gene expression, cell wall biosynthesis, and sucrose accumulation during

development of Sorghum bicolor, Plant J. 88 (2016) 662-680.

32
[64] S. Gutjahr et al., Grain, sugar and biomass accumulation in photoperiod-sensitive

sorghums. II. Biochemical processes at internode level and interaction with phenology,

Funct. Plant Biol. 40 (2013) 355-368.

[65] R. Wendler, R. Veith, J. Dancer, M. Stitt, E. Komor, Sucrose storage in cell suspension

cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation,

Planta 183 (1991) 31-39.

[66] A. Krapp, B. Hofmann, C. Schäfer, M. Stitt, Regulation of the expression of rbcS and

other photosynthetic genes by carbohydrates: a mechanism for the ‘sink regulation’ of

photosynthesis?, The Plant J. 3 (1993) 817-828.

[67] W.L. Rooney, J. Blumenthal, B. Bean, J.E. Mullet, Designing sorghum as a dedicated

bioenergy feedstock, Biofuels, Bioproducts and Biorefining 1 (2007) 147-157.

[68] J.I. Bonner et al., Opportunities for energy crop production based on subfield scale

distribution of profitability, Energies 7 (2014) 6509-6526.

[69] N.E. Borlaug, Sixty-two years of fighting hunger: personal recollections, Euphytica 157

(2007) 287-297.

33
Figure Legends

Figure 1: Comparison of grain sorghum and high-biomass sorghum.

Photographs of short early flowering grain sorghum (left) and high-biomass sorghum

(right) after ~110 days of a 180-day growing season. The shorter stature of grain sorghum

is due to mutations in Dw1 and Dw3 and floral initiation in ~60 days. The high-biomass

sorghum hybrids shown will undergo floral transition after ~150 days, reach a height of 4-

5 m and accumulate more than twice as much biomass as grain sorghum during the

growing season.

Figure 2: Pathway regulating sorghum flowering time in response to photoperiod.

The C4 grass genes CN8, CN12 and CN15, homologs of FT, are sources of leaf-derived

florigen that moves to the shoot apical meristem (SAM) activating floral initiation.

Expression of the floral-promoting CN-genes is activated by CONSTANS (CO) and EHD1.

PRR37, GHD7 and CDF1 encode repressors of CONSTANS, EHD1 and CN-gene

expression/activity. Light signaling mediated by phytochrome B (encoded by PHYB) and

phytochrome C (encoded by PHYC) and output from the circadian clock regulate expression

of PRR37, GHD7, and CDF1 such that floral initiation is repressed in long days but not in

short days.

Figure 3. High-biomass C4 grass stem morphology.

Stems of high-biomass C4 grasses are comprised of nodes and internodes that are produced

by the shoot apical meristem during vegetative growth. The length of high-biomass

sorghum internodes is determined by environmental conditions and can be reduced by

34
mutations in Dw1, Dw2 or Dw3. Solid stems comprised of vascular bundles, pith

parenchyma, fiber cells and epidermal cells can accumulate high levels of sugars following

floral induction.

35
FIGURES: High-Biomass C4 Grasses – Filling the Yield Gap

Figure 1

Figure 2

Figure 3
Table 1: Genes Regulating Sorghum Flowering Time in Response to Photoperiod

Gene name Gene Function

CN8, CN12, CN15 Sources of florigen, homologs of FT

CONSTANS (CO) Activator of EHD1, CN8, CN12 expression

EHD1 Activator of CN8, CN12 expression

PRR37 Repressor of CONSTANS, EHD1, CN8, CN12

CDF1 Proposed repressor of CONSTANS, CN8, CN12

PHYB (phyB) Phytochrome B, red light photoreceptor

PHYC (phyC) Phytochrome C, red light photoreceptor

36