Pullulanase Activity is Associated with Formation of Vitreous Endosperm in Quality Protein Maize

Wu, Hao ; Clay, Kasi ; Thompson, Stephanie S. ; Love, Sterling ; Gibbon, Bryan C. Department of Biology, Baylor University; One Bear Place 97388; Waco, TX, 76798

Abstract Results and Discussion

Introduction

AcknowledgementsThis work was supported by AFRI Competitive Grant no. 2010-65115-20375 from USDA-NIFA and by the URSA small grant program of Baylor University .

The opaque2 (o2) mutation of maize increases lysine and tryptophan content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. Four starch biosynthesis genes, SSIIa, SSIIb, SSIII and Zpu1, have been discovered to have unique alleles in mo2 lines; therefore these genes may play a role in formation of vitreous endosperm. qPCR analysis of recombinant inbred lines (RILs) derived from a cross of QPM and soft o2 lines showed a significant increase in expression of the QPM-derived ZpuI allele. Quantitative enzyme activity assays showed that QPM lines had higher pullulanase activity than o2 and wild type. Furthermore, pullulanase activity was positively correlated with kernel vitreousness in the RILs. Differential scanning calorimetry showed that the thermal properties of starch from the RILs correlated well with the presence of the QPM-derived allele of Zpu1, which had decreased onset and peak endotherm temperatures while total enthalpy of gelatinization was unchanged. Pullulanase activity was negatively correlated with the onset and peak endotherm temperatures but was not correlated with enthalpy. Additionally, pullulanase activity was negatively correlated with the sensitivity of starch granules to digestion by amylase. From these data, we hypothesize that pullulanase is one of the factors that influence amylopectin branch length and crystallinity, which in turn affect the formation of vitreous endosperm in QPM.

The Problem with opaque2 The endosperm is soft and chalky

More vulnerable to insect damage More prone to breakage during harvest

Yield is significantly reduced

Suppression of opaque2 (Modified opaque 2, mo2)

Segregating ear of recombinant inbred lines (RILs) from a soft o2 x QPM (mo2) cross demonstrates that modification is variable

The phenotypes range from completely opaque (o2) to highly translucent (mo2)

mo2 kernels have a hard, vitreous phenotype with increased level of 27-kDa gamma-zeins and some non-zein proteins, as well as relatively high lysine content.

The opaque2 (o2) mutant has small protein bodies and these protein bodies are essentially devoid of lysine. Thus the opaque2 mutant has higher lysine and tryptophan content and is nutritionally superior to normal maize seed.

Wild type

opaque2

The opaque2 Mutation

Prior research indicated that there was a change in the structure of starch granules in modified o2 lines that resulted in adhesions between starch granules that were not observed in wild type or opaque2 mutants. Our current research is focused on the genetic and biochemical changes that have occurred in the modified opaque2 lines to restore hard kernel phenotypes, while maintaining superior amino acid balance.

SEM of CM105 WT SEM of CM105 o2 SEM of CM105 mo2

Expression levels of Zpu1 gene in parents (right) and RILs (left)

In RILs derived from QPM, the Zpu1 allele exhibited significantly higher expression than that derived from o2. The parental data only showed a slightly higher expression level in K0326Y than W64Ao2. However the trend was consistent with that of increasing pullulanase activity. The pullulanase activity of QPM was significantly higher compared to o2, which was significantly higher than wildtype.

Pullulanase activity influences the kernel vitreousness, sensitivity of starch to amylase, and

thermal properties of starch

Thermal Properties of Starch

Differential scanning calorimetry (DSC) analysis of W64Ao2, K0326Y and zpu1 mutant showed that there was no significant difference between o2 and QPM parental lines, but the QPM lines had lower enthalpy, indicating that QPM starch granules might have lower overall crystallinity; also compared with wildtype, the zpu1 mutant had higher onset and maximum temperature, but had similar enthalpy, indicating the similar crystallinity. In addition, the DSC study of RILs derived from a cross of a QPM and o2 lines showed that the thermal properties of the starch correlated well with the presence of two of the QPM-derived alleles. For Zpu1, the onset and peak endotherm temperatures for lines homozygous for the QPM allele were lower than for the o2 allele, whereas total enthalpy of gelatinization was not significantly changed. For SSIII, the onset and peak endothermic temperatures significantly increased, while total enthalpy of gelatinization became significantly lower. The data suggest that the onset and maximum temperature can be affected by both Zpu1 and SSIII; while enthalpy might be influenced by SSIII, rather than Zpu1. Also, the average length of glucan chains could be shorter in lines expressing the QPM Zpu1 allele, while the overall granule crystallinity of both lines expressing QPM and o2 Zpu1 alleles could be similar.

69.8

70

70.2

70.4

70.6

70.8

71

Ons

et T

empe

ratu

re (

o C)

*

72.8

73

73.2

73.4

73.6

73.8

74

74.2

74.4

Max

imum

Tem

pera

ture

(oC)

*

6

7

8

9

10

11

12

Enth

alpy

(J/g

)

69

69.5

70

70.5

71

71.5

Ons

et T

empe

ratu

re (o

C)

*

72.8

73

73.2

73.4

73.6

73.8

74

74.2

74.4

Max

imum

Tem

pera

ture

(o C

)

*

6

7

8

9

10

11

12

13

Enth

alpy

(J/g

)

*

RILZpu1

RIL SSIII

Onset Temperatures Maximum Temperature Enthalpy

Parents

zpu1 mutant

o2 QPM o2 QPM o2 QPM

o2 QPM o2 QPM o2 QPM

64

65

66

67

68

69

70

71

72

W64Ao2 K0326Y (QPM)

Ons

et T

empe

ratu

re (o

C)

67

68

69

70

71

72

73

74

75

76

W64Ao2 K0326Y (QPM)

Max

imum

Tem

pera

ture

(oC)

0

2

4

6

8

10

12

14

16

18

W64Ao2 K0326Y (QPM)

Enth

alpy

（J/

g)

*

64

65

66

67

68

69

70

71

72

W64A+ zpu1

Ons

et T

empe

ratu

re (o

C)

*

67

68

69

70

71

72

73

74

75

76

W64A+ zpu1

Max

imum

Tem

pera

ture

(oC) *

0

2

4

6

8

10

12

14

W64A+ zpu1

Enth

alpy

（J/

g)

Note: * indicates p < 0.05 by two tailed t-test.

W64A+ W64Ao2 K0326Y0

0.05

0.1

0.15

0.2

0.25

Pullu

lana

se A

ctivi

ty

A

B

C

Pullulanase activity of W64A+, W64Ao2 and

K0326y

0.100 0.150 0.200 0.250 0.300 0.350 0.4000

20

40

60

80

100

120

140

160

180

Pullulanase activity

Vitr

eous

ness

0.100 0.150 0.200 0.250 0.300 0.350 0.4000.25

0.27

0.29

0.31

0.33

0.35

0.37

0.39

Pullulanase Activity

Amyl

ase

sens

itivi

ty

68.5 69 69.5 70 70.5 71 71.5 720.10

0.15

0.20

0.25

0.30

0.35

Onset Temperature (oC)

Pullu

lana

se A

ctivi

ty

8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 130.10

0.15

0.20

0.25

0.30

0.35

Enthalpy (J/g)

72 72.5 73 73.5 74 74.5 750.10

0.15

0.20

0.25

0.30

0.35

Maximum Temperature (oC)

Pullulanase activity was significantly correlated with the kernel vitreousness, amylase sensitivity, and onset/maximum temperatures, suggesting that as a starch debranching enzyme, pullulanase might influence the fine structure of starch, which in turn affect the kernel vitreousness and properties of starch granules.