Physiological and Molecular

Basis of Ethephon to

Enhance Sucrose Content

in Sugarcane

Litao Yang Guangxi University

liyr@gxu.edu.cn

2014. 02. 16

Sugar industry in Guangxi

In 2012/2013 milling season, Guangxi

grew ~1 million ha of sugarcane, and

produced 66.36 million tons of millable

cane and 7.95 million tons of white sugar.

The average cane yield is ~70.0 tons/ha.

Milling season: November ~ April

Socrose content (%): ~ 14.5%

Sucrose in Sugarcane

Sucrose accumulate in parenchyma tissue of

sugarcane stalk, up to 60% of its dry weight or

25% (w/w) of its fresh weight

Sucrose yield (t ha-1) =

cane yield (t ha-1) × recoverable sucrose (%) ÷ 100

Recoverable sucrose (%) =

[ sucrose % — (Brix % — sucrose %) ÷ 2 ] × extraction %

Major Factors Affecting Sucrose Content

Species and Varieties

Plant age: sucrose concentration progressively

increases towards the base of the stalk

Environment:

light, temperature, water, soil nutrient, weeds,

pests, diseases, farming conditions

Increase Sucrose Content in Sugarcane

Breeding programs: New Varieties

Physiological approaches:

Leaf photosynthesis (source)

Enzymes involved in sucrose synthesis;

Sucrose transfer (phloem loading and transport

to the ripening stalk);

Deposition in stalk (carbon partitioning within

the stalk/vacuoles, and also rate of sucrose

remobilization to support vegetative growth )

(cited from Wang et al., 2013)

UDP-Glu

Plant Hormone: Ethylene

Plant hormones including ABA, ethylene and

GA were involved in the control of sucrose

levels in plant cells.

Ethylene is a gaseous plant hormone that

influences a number of processes in plants

such as seed germination, abscission,

senescence, fruit ripening, response to

stress, and growth.

Ethephon is an ethylene releasing compound

Molecular biological approaches

Increase Sucrose Content in Sugarcane

To understand the synthesis and accumulation

of sucrose, and the regulation mechanisms of

sucrose accumulation in the stalk;

To identify and characterize the genes involved

in sucrose metabolism and regulation;

To manipulate sugarcane sucrose contents by

transgenesis.

Sugarcane genetypes with

different sucrose contents

Genomic and proteomic analysis

Gene functional analysis

Physiological approaches in

sucrose metabolism and

accumulation

Molecular regulation network

The mechanism of sucrose synthesis, accumulation and

regulation

Ethylene induced

Environmental conditions

Relationship between sucrose

content and characterization of

physiology and biochemistry

Objectives

1. The effect of ethylene on sucrose content.

2. The physiological mechanism of ethylene

on increase sucrose content.

3. Isolation and functional analysis of

sugarcane genes associated with sucrose

content.

4. Understanding the physiological and molecular

mechanism of sucrose synthesis, accumulation

and regulation in sugarcane.

1. Effect of ethephon on sucrose content in plot

experiment (small area)

Effect of ethephon on increasing sucrose content in mature (internode 8) and

immature (internode 4) internodes in 3 sugarcane varieties (ROC16, early

muturing; GT11, early-intermediate maturing; GT15, intermediate-late

maturing) through foliage sprayed (400 mg/L).

Ethephon significantly decreased the reduceing sugar

content in juice of mature (internode 8) and inmature

(internode 4) internodes for 3 varieties

2. Effects of ethephon on sucrose content and yield in

large area of sugarcane

The experiment was conducted at 3 state farms (Jingguang, Liangqi

and Lutang) in total of 2400 ha of sugarcane (plant and ratoon).

Increase sucrose: ~ 1.36%

Increase cane yield: 4.02~6.33%

Ethephon increased green leaf numbers and chlorophyll contents

A: Jin-Guang B: Liang-Qi, C: Lu-Tang D: chlorophyll contents

SS:sucrose synthase; SPS: sucrose phosphate synthase;

NI:neutral invertase; AI: acid invertase

Sugarcane seedling (ROC16) was foliage sprayed with 200 mg/L of ethephon

3. Effects of ethephon on enzyme activities involved in

sucrose metabolism

4. Effects of ethephon on differential gene expression

Differential gene expression in

sugarcane leaf with ethephon

application and control by cDNA-

AFLP analysis.

Ethephon: 2, 4, 6; Control: 1, 3, 5

Partial differential TDFs

Notes: 1TDF (Transcript derived fragment) codices corresponding to

polymorphism band in polyacrilamide gel; 2Results of BLAST, “--” represents no

homology sequences; 3Identified protein, “--”represents no identified protein; 4

Expect value.

The results indicated that ethephon could regulate

the expression of genes related to the primary

metabolism, resistance to disease and stress in

sugarcane.

The differentially expressed genes include:

chitinase I (CHI),

glutathione S-transferase (GST),

auxin-responsice protein (ARP),

light harvesting chlorophyll a/b-binding protein (LHC),

nuclear binding protein (NBP) ,

a set of unknown genes

5. Cloning and expression analysis of

sugarcane sucrose relative genes

(1) Ethylene receptor (Sc-ERS) gene

(2) Sucrose phosphate synthase (SofSPSA and SofSPSB) gene

(3) Soluble acid invertase (SoSAI1) gene

Expression analysis of SoSAI1 in different organs and growth stages of sugarcane.

A: Elongation stage; B: Technical maturing stage; C: Physiological maturing stage;

D: Physiological matured stage 1-3: leaves at immature, maturing and matured,

respectively; 4-6: internodes of immature, maturing and matured; 7: rachis; 8:

inflorescence.

(4) alkaline/neutral invertase (SoNIN1)

Expression of SoNIN1 gene in different sugarcane organs. 1–7: internodes of +1, +6, +11, +16, +21, +26, +31, respectively;

8-11: leaves of top leaf roll, +1, +3, +6, respectively;

12: rachis; 13: inflorescence; 14: bud.

1. Biological funtional analysis of sugarcane

sucrose relative genes (localization and

transgenetics)

2. Genomic and proteomic analysis of

sugarcane genetypes with different sucrose

contents

3. Set up the network of sucrose synthesis,

accumulation and regulation

Further Research

Acknowledgements

PI: Prof. & Dr. Yang-rui LI,

GXAAS: Drs. Bao-Qing Zhang, Yuan-Wen Wei, Dong-Liang Huang,

Prof. Qiu-zhen Zhu

GXU: Prof. Ai-Qin Wang,

Ph.D. students: Jun-Qi Niu

State Farms: Wu-jia Cen, Zhi-biao LanN, Hua-ming Guang,

Zhong-Bo Huang

Funding:

National Science and Technology Program

National Nature Science Foundation

Guangxi R & D Research Program

Thank you!