Universidad de Sonora

ISSN: 1665-1456

Volume XXV, Issue 2

Journal of biological and health sciences

http://biotecnia.unison.mx

*Corresponding author: Jose A. Lopez Valenzuela

e-mail: jalopezvla@uas.edu.mx

Received: December 1, 2022

Accepted: February 5, 2023

Mechanisms associated with endosperm modication

in quality protein maize

Mecanismos asociados con la modicación del endospermo en maíz de calidad proteínica

David Guillermo Gonzalez-Nuñez, Karen Virginia Pineda-Hidalgo, Nancy Yareli Salazar-Salas, Jose Angel Lopez-

Valenzuela*

Faculty of Chemical-Biological Sciences, Autonomous University of Sinaloa, University City, Av. Americas y Josefa Ortiz de

Dominguez S/N, CP , Culiacan, Sinaloa, Mexico.; davidggn@gmail.com, kvpineda@uas.edu.mx, nancy.salazar@uas.

edu.mx, jalopezvla@uas.edu.mx

ABSTRACT

Quality protein maize (QPM) combines the protein quality of

the opaque-2 (o2) mutant with a vitreous endosperm. These

characteristics have allowed breeding programs worldwide

to produce QPM genotypes that help alleviate malnutrition

of people in developing countries from Africa, Asia and

Latin America with a cereal-based diet. However, the deve-

lopment of these materials has been inecient due to the

limited knowledge about the molecular basis of the soft o2

endosperm conversion into a vitreous phenotype in QPM.

This conversion has been associated with an increase in small

protein bodies rich in 27 kDa γ-zein, the synthesis of starch

with a higher proportion of amylose and short-intermediate

amylopectin chain branches that favors the compaction of

the starch granules, as well as alterations in the amyloplast

envelope that favors the interaction between starch granules

and protein bodies. Additional studies about the mecha-

nisms involved in the modication of the endosperm in QPM

will contribute to produce materials with good agronomic

characteristics and protein quality.

Keywords: Zea mays L.; endosperm modication; starch;

zeins

RESUMEN

El maíz de calidad proteínica (MCP) combina la calidad

proteínica de la mutante opaco-2 (o2) con un endospermo

vítreo. Estas características han permitido a los programas

de mejoramiento alrededor del mundo producir genotipos

MCP que ayudan a aliviar la malnutrición de la gente en

países en desarrollo de África, Asia y América Latina con

una dieta basada en cereales. Sin embargo, el desarrollo de

estos materiales ha sido poco eciente debido al limitado

conocimiento acerca de las bases moleculares de la con-

versión del endospermo suave o2 en un fenotipo vítreo en

MCP. Esta conversión se ha asociado con el incremento en

cuerpos proteínicos pequeños ricos en γ-zeína de 27 kDa, la

síntesis de almidón con una mayor proporción de amilosa y

ramicaciones de amilopectina cortas-intermedias que favo-

rece la compactación de los gránulos de almidón, así como

alteraciones en la envoltura de los amiloplastos que favorece

la interacción entre gránulos de almidón y cuerpos proteíni-

cos. Estudios adicionales sobre los mecanismos involucrados

en la modicación del endospermo en MCP contribuirán a

producir materiales con buenas características agronómicas

y buena calidad proteínica.

Palabras clave: Zea mays L., modicación del endospermo,

almidón, zeínas

INTRODUCTION

Maize (Zea mays L.) is the most important cereal with a global

production of 1,162 million tons in 2020 (FAOSTAT, 2022).

This cereal has a great social impact in developing countries

where it is the main food staple. However, the most abun-

dant proteins of maize (prolamins or zeins) are decient in

the essential amino acids lysine and tryptophan aecting the

nutritional quality of the grain.

Mertz et al. (1964) found that the opaque-2 (o2) muta-

tion (Figure 1) almost doubles the lysine content in maize

endosperm and improves the protein quality, but the use of

this mutant in breeding programs was limited due to its poor

agronomic performance associated with the opaque/soft

endosperm and low seed density. Some years later, Paez et

al. (1969) found that some segregating S2 lines derived from

opaque S1 parents showed modied o2 kernels (50% translu-

cent or vitreous) whose lysine content was not dierent from

the opaque kernels. Therefore, selection for hard endosperm

was started. Researchers from the International Maize and

Wheat Improvement Center (CIMMYT) in Mexico (Villegas et

al., 1992) and the University of Natal in South Africa (Gevers

and Lake, 1992), developed a modied o2 mutant or quality

protein maize (QPM) (Figure 1) by recurrent backcrossing.

This process required the simultaneous selection of kernels

with normal texture and enhanced levels of essential amino

acids. Thus, QPM combines the protein quality of o2 with a vi-

treous endosperm and has better agronomic characteristics.

The QPM materials developed by the CIMMYT maize

breeding program have been used worldwide as donors of

o2 modiers to produce lines and hybrids adapted to each

region. However, conventional QPM breeding involves the

introgression of o2 into a local adapted genotype that is sub-

DOI: 10.18633/biotecnia.v25i2.1905

Volume XXV, Issue 2

Gonzalez-Nuñez et al: Biotecnia / XXV (2): 79-89 (2023)

sequently used to pollinate a modier donor, a process that

requires several generations of backcrossing and self-crossing

and the monitoring of high levels of lysine and tryptophan,

the recessive o2 mutant allele, and the modiers. This lengthy

and laborious strategy can take more than six years. The use

of molecular markers has facilitated QPM breeding but this

process could be more ecient if the mechanisms involved

in the conversion of the soft o2 endosperm into a vitreous

phenotype were understood (Gibbon and Larkins, 2005). The

main mechanisms proposed for this conversion include an

increase in the accumulation of small protein bodies enri-

ched in 27 kDa γ-zein (Wu et al., 2010), the alteration of the

structure and composition of starch due to the increase in the

proportion of amylose and short-intermediate amylopectin

chain branches (Gibbon et al., 2003; Salazar-Salas et al., 2014)

and the loss of the amyloplast envelope due to a reduction

in non-polar carotenoids (Wang et al., 2020). The aim of the

present review is to provide the current advances about the

molecular mechanisms associated with the modication of

the vitreous endosperm in QPM.

MAIZE KERNEL COMPOSITION

The major components of the maize kernel are starch (64 -

78 %) and proteins (8 - 15 %). Starch is mainly found in the

endosperm while proteins are more abundant in the germ.

The remaining components of the grain consist of lipids (4.0

- 4.9 %), ashes (1 - 3 %) and ber (1 - 2 %) (Serna-Saldivar,

2019). Starch and proteins inuence the physicochemical

and structural characteristics of the kernel, highlighting the

importance of these components.

Starch biosynthesis

Starch is the main carbon reserve in cereals (70 - 80 %) and

is mainly responsible for their energetic and economic va-

lue. It is formed by two homopolysaccharides: amylose, an

essentially linear molecule formed by glucose units linked

by α-(1,4) glycosidic bonds, and amylopectin, a molecule for-

med by linear portions of glucose linked by α-(1,4) glycosidic

bonds and ramications linked by α-(1,6) bonds (Pster and

Zeeman, 2016).

In cereals, starch is mainly found in endosperm cells

forming granules and its biosynthesis is carried out by the

coordinated action of multiple enzymes: ADP-Glucose pyro-

phosphorylase (AGPase), starch synthases (GBSS, SS), starch

branching enzymes (SBE) and starch debranching enzymes

(DBE) (Figure 2) (Comparot-Moss and Denyer, 2009). This

process begins with the enzyme AGPase that produces

ADP-glucose in the cytosol, which is transported into the

plastid and serves as a substrate for starch synthase enzy-

mes (Pster and Zeeman, 2016). The granule bound starch

synthase I (GBSSI) is encoded by the waxy locus in cereals and

is responsible for the elongation of amylose, being essential

within the granule matrix. The soluble starch synthases (SSI,

SSII, SSIII and SSIV) are exclusively involved in the synthesis

of amylopectin chains and are associated with the starch

granules. Genetic and biochemical studies indicate that each

SS isoform has dierent properties and a dierent role in

amylopectin synthesis. The starch branching enzyme (SBE)

generates α-(1,6) glycosidic bonds after breaking the α-(1,4)

bond and transferring the chain to the C6 from a glucose

residue of another chain, forming the branched structure of

the amylopectin molecule (Huang et al., 2021). Two isoforms

of branching enzymes are expressed in the endosperm of

cereals, branching enzyme I (SBEI) and branching enzyme II

(SBEII), which dier in the length of the transferred glucan

chain and substrate specicity; SBEI shows greater anity for

amylose and transfers longer chains than SBEII, which trans-

fers shorter chains and has greater anity for amylopectin

(Sawada et al., 2018).

The starch debranching enzyme (DBE) catalyzes the

hydrolysis of glycosidic bonds α-(1,6). In higher plants the-

Opaque endosperm

Vitreous endosperm

kDa

48.5

34.4

27.2

17.1

Vitreous Opaque

γ-zein 27 kDa

α-zein 22 kDa

α-zein 19 kDa

γ-zein 50 kDa

γ-zein 16 kDa

Figure 1. Zein proles and endosperm phenotype of vitreous (QPM) and

opaque maize genotypes. (A) SDS-PAGE separation of zein proteins. The

molecular weight marker (kDa) is shown on the left and the dierent zein

sub-fractions are indicate on the right. A greater abundance of 27-kDa γ-zein

can be observed in the endosperm of vitreous lines compared to the opaque

ones. Adapted from Vega-Alvarez et al. (2022). (B) Cross section of K0326Y

(vitreous) and W64Ao2 (opaque) maize kernels viewed under white light.

white light.

Figura 1. Perles de zeínas y fenotipo del endospermo en genotipos de

maíz vítreos (QPM) y opacos. (A) Separación por SDS-PAGE de proteínas

zeínas. El marcador de peso molecular (kDa) se muestra a la izquierda y

las diferentes subfracciones de zeínas se indican a la derecha. Se puede

observar una mayor abundancia de γ-zeína 27 kDa en el endospermo de

líneas vítreas comparado con las opacas. Adaptado de Vega-Alvarez et al.

(2022). (B) Sección transversal de granos de maíz de las líneas K0326Y (vitrea)

y W64Ao2 (opaca) vistos bajo luz blanca. (C) Granos enteros de maíz de las

líneas K0326Y (vitrea) y W64Ao2 (opaca) vistos bajo luz blanca.

Volume XXV, Issue 2

Gonzalez-Nuñez et al: Mechanisms associated with endosperm modication / XXV (2): 79-89 (2023)

re are two types of DBE and they are dened according to

the specicity of their substrate: debranching enzymes of

the isoamylase type and the pullulanase type. Isoamylase

breaks down amylopectin and phytoglycogen branches,

while pullulanase acts on pullulans and amylopectin but not

phytoglycogen (Robyt, 2009).

Synthesis of zeins

The most abundant storage proteins in maize kernels are

prolamins (soluble in alcohol) or zeins (50-55%), followed

by glutelins (soluble in alkaline solutions) (35-40%), while

albumins (soluble in water) and globulins (soluble in saline

solutions) account for less than 10% of the total proteins

(Larkins, 2019; Sethi et al., 2021). Because zeins are the major

proteins in the kernel, total proteins are typically divided into

zeins and non-zeins. Zeins are synthesized by membrane-de-

limited polyribosomes and transported within the lumen of

the rough endoplasmic reticulum, where they assemble into

protein bodies (Lending and Larkins, 1989). Protein bodies

are small spherical structures made up of a protein matrix

surrounded by a simple membrane; they contain at least four

types of zeins (α-, β-, γ-, δ-), which are classied based on

their solubility and structural similarities as α-zeins (22 and

19 kDa), β-zeins (14 and 16 kDa), γ-zeins (16, 27 and 50 kDa)

and δ-zeins (10 kDa) (Figure 1A) (Coleman and Larkins, 1999).

Immunolocalization studies showed that γ- and β-zeins are

generally located in the peripheral region of protein bodies,

while α-zeins are located in the internal region of these struc-

tures (Lending and Larkins, 1989).

The o2 mutant has a defective transcription factor that

regulates the expression of α-zeins and depending on the

genetic background the content of these proteins can be

reduced more than 50% (Figure 1A) (Kodrzycki et al., 1989).

Zeins are decient in some essential amino acids (lysine and

tryptophan) and the reduced synthesis of these proteins in

o2 results in higher levels of more nutritionally balanced non-

zeins (Lopez-Valenzuela et al., 2004) and free amino acids in

the endosperm (Pineda-Hidalgo et al., 2011). The use of RNA

interference (RNAi) to block the expression of α-zeins has

also shown to increase the levels of lysine and tryptophan

in maize endosperm (Huang et al., 2006), which avoids the

negative characteristics and limitations of the recessive o2

mutant. The Cas9/CRISPR technology represents another

biotechnological alternative to reduce or remove zein gene

expression as a strategy to increase the levels of proteins with

a better balance of essential amino acids (Jiang et al., 2022).

QUALITY PROTEIN MAIZE AND ITS

IMPORTANCE IN HUMAN NUTRITION

Malnutrition is a problem that aects more than 828 million

people worldwide, 98% of which are from developing cou-

ntries, mainly Africa, Asia, Latin America and the Caribbean,

and includes 150 million children (FAOSTAT, 2022). People

from developing countries with a cereal-based diet have a

high risk of protein and lysine deciency (Muleya et al., 2022),

although this deciency can occur in any people with a diet

based on cereals. Several investigations have documented

the benets of QPM in human nutrition, highlighting its po-

tential to mitigate problems associated with protein-energy

deciency in children under 5 years of age, the elderly and

pregnant women, considered the most vulnerable groups

(Hossain et al., 2019).

The consumption of QPM instead of normal maize, in-

creased 12 - 15% the weight growth rate in infants and young

children, with mild to moderate undernutrition (Akalu et al.,

2010; Gunaratna et al., 2010). QPM has also a higher content

of phenylalanine and isoleucine, suggesting it can be inclu-

ded in the family diet to reduce the risk of inadequate protein

intake (Gunaratna et al., 2019). Tortillas from nixtamalized and

extruded QPM ours showed higher nutritional indicators (C-

PER, protein digestibility, PER, NPR, PDCAAS) than those from

normal maize, suggesting they may have a positive eect

on the nutritional status of people from countries where

these products are widely consumed (Gutiérrez-Dorado et

al., 2008). Desalegn et al. (2015) showed that QPM based

complementary foods have good sensory acceptability and

can help meet the minimum recommended daily dose of

energy (370 kcal) and protein (10.9 g) for children aged 6 -

36 months, as well as two thirds of the recommended iron

and zinc daily dose and up to 50 % of vitamin A. The supple-

mentation of malnourished young children (4 - 6 years old)

with QPM-based biscuits reduced the percentage of anemic

subjects from 63.3 % to 16.6 % and the prevalence of severe

underweight from 23.3 % to 0 % (Grover et al., 2020).

In recognition of the great potential of QPM to improve

human nutrition in poor countries where maize is a staple

food, Dr. Surinder K. Vasal and Dr. Evangelina Villegas from

Figure 2. Starch biosynthesis pathway in the endosperm of cereals.

The cytosol and plastid are indicated. Enzymes are indicated in italics:

Susy, sucrose synthase; UGPase, UDP glucose pyrophosphorylase; PGM,

phosphoglucomutase; FK, fructokinase; PGI, phosphoglucose isomerase;

PPiase, pyrophosphatase; AGPase, ADP-glucose pyrophosphorylase; GBSSI,

granule bound starch synthase; SS, starch synthase; SBE, starch branching

enzyme. Adapted from Comparot-Moss and Denyer (2009).

Figura 2. Ruta de síntesis de almidón en el endospermo de cereales. Se

indican el citosol y el plástido. Las enzimas están indicadas en itálicas:

Susy, sacarosa sintasa; UGPasa, UDP glucosa pirofosforilasa; PGM,

fosfoglucomutasa; AGPasa, ADP-glucosa pirofosforilasa; GBSSI, almidón

sintasa unida al gránulo; SS, almidón sintasa; SBE, enzyma ramicadora de

almidón. Adaptada de Comparot-Moss and Denyer (2009).

Volume XXV, Issue 2

Gonzalez-Nuñez et al: Biotecnia / XXV (2): 79-89 (2023)

CIMMYT were awarded with the World Food Prize in 2000

(Cordova, 2001).

ADVANCES IN THE DEVELOPMENT OF QPM

Despite the QPM nutritional value and agronomic perfor-

mance, the cultivation and adoption of these materials

on a large scale has not been achieved, mainly due to the

low availability of genetically diverse and competitive QPM

hybrids compared to non-QPM / normal hybrids, the lack

of information about their health benets and government

incentives (Hossain et al., 2018; Maqbool et al., 2021). Never-

theless, breeding programs have been implemented around

the world with the purpose of producing new and better

QPM genotypes; they have mainly used QPM donors from

CIMMYT in Mexico (Cordova, 2001; Vivek et al., 2008). Until

2015, more than 167 QPM varieties were released worldwi-

de, 53 % in Africa, 25 % in Latin America and 22 % in Asia

(Twumasi-Afriyie et al., 2016). Some of these QPM genotypes

are listed in Table 1. Conventional breeding strategies such as

recurrent selection were initially used for QPM development,

but in the last decades a widely used strategy to develop

these materials is molecular marker-assisted breeding and

most of the studies have used SSR markers (e.g. phi 057, phi

112 and umc 1066) located within the o2 gene on the short

arm of chromosome 7 (www.maizegdb.org) (Maqbool et al.,

2021). Some examples include the QPM version of the line V25

derived from the cross V25 × CML176 (QPM), which shows an

increase in tryptophan content (0.85 %) and maintains a hard

endosperm (Babu et al., 2005), as well as Vivek QPM-9 (VQL

1 × VQL 2) that contains 41 % more tryptophan and 30 %

more lysine than the original hybrid (Vivek Hybrid-9) (Table

1) (Gupta et al., 2013).

Other researchers have focused on developing new

QPM genotypes with certain agronomic and gastronomic

characteristics, but always seeking to maintain the nutritional

quality of o2. For instance, the Zhao OP-6 /o2o2 corn was ge-

nerated by introducing the o2 allele into the Zhao OP-6 waxy

corn to produce a waxy QPM line intended for the Chinese

market, where waxy corn is widely used in food processing

because of its high viscosity and digestibility (Zhou et al.,

2016). Quality Protein Popcorn (QPP) was developed recently,

which showed a higher lysine content compared to its parent

elite line and maintained its bursting capacity, demonstrating

the potential use of QPM for the production of grains with

specic functional characteristics and good protein quality

(Ren et al., 2018). Since most of the eorts to develop QPM

have been based on the use of molecular markers that co-

inherit with the o2 phenotype, improving the understanding

of the molecular basis of endosperm modication could help

to develop these materials more eciently.

MECHANISMS ASSOCIATED WITH ENDOSPERM

MODIFICATION IN QPM

Increased accumulation of γ-zein proteins

One of the rst biochemical changes observed in QPM

genotypes was an increase in γ-zein content (2 to 5 times)

compared to o2 (Figure 1A) and normal maize (Wallace et al.,

1990). Immunolocalization studies suggested that γ-zein ini-

tiates the formation of protein bodies (Lending and Larkins,

1989), which is supported by the fact that inhibition of the

27 kDa γ-zein encoding gene reduces signicantly the num-

ber of protein bodies, while the inhibition of 19 and 22 kDa

α-zeins decreases the diameter of these structures (Guo et al.,

2013). The γ-zeins are highly linked with disulde bonds and

it has been hypothesized that the covalent bonds of γ-zeins

and other cysteine rich proteins promote the formation of a

protein network around starch granules (Lopes and Larkins,

1991). Therefore, it has been proposed that an increase in the

number of protein bodies and their compaction among the

starch granules is, at least partially, responsible for the mo-

dication of the endosperm (Figure 3A). A genetic analysis

using recombinant inbred lines (RIL) derived from the cross

of Pool 33 (QPM) and W64Ao2 (soft endosperm), identied

two loci associated with endosperm modication; these loci

were located on the long arm of chromosome 7, one near

the centromere linked to a 27 kDa γ-zein locus and the other

near the telomere (Lopes et al., 1995). Holding et al. (2008)

identied 7 loci associated with o2 modier genes (mo2) in

an F2 progeny from the cross of K0326Y-QPM and W64Ao2;

two major loci located on chromosomes 7 and 9 explained

40 % of the phenotypic variation and were linked to 27 kDa

γ-zein and starch synthesis genes, respectively. Holding et al.

(2011) developed RIL from the F2 progeny and the kernels

were characterized for vitreousness, density and hardness;

genetic mapping with these RIL identied loci on chromo-

somes 1, 7 and 9, conrming their linkage with γ-zein and

starch biosynthesis genes.

Several studies have focused on the 27 kDa γ-zein locus

to understand the mechanism of o2 modier genes. There is

more than one gene for 27 kDa γ-zein in this locus, which are

assigned depending on the genotype and are called A and

B, and together form the AB locus (Das et al., 1990). The AB

locus of γ-zeins is not stable and can be rearranged to form

A and B, called rA and rB, respectively (Das et al., 1990; Das

et al., 1991). Because all known QPM lines contain the A and

B genes, and all F2 progeny from a cross of a QPM and an

o2 mutant contain the AB locus, it has been hypothesized

that the AB locus is necessary for endosperm modication,

although it is also emphasized that this locus by itself is not

sucient to achieve such modication (Lopes et al., 1995).

Although the molecular mechanism of the mo2 genes

is not fully understood, evidence suggests that a greater

stability of the γ-zein mRNA and/or a higher transcription

rate may be responsible for a larger accumulation of the 27

kDa γ-zein protein in modied o2 genotypes. Geetha et al.

(1991) suggested that the role of mo2 genes is to increase the

stability of γ-zein mRNA and protein synthesis. Or et al. (1993)

suggested that the stability of the A gene mRNA initiates en-

hanced 27 kDa γ-zein protein synthesis. Burnett and Larkins

(1999) found that the A:B ratio of γ-zein mRNA in mo2 endos-

perms was more than 40:1, compared to a 1:1 ratio for normal

maize and 3:1 for o2, indicating that these relationships can

result from dierent transcription rates of the genes A and B.

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Table 1. Development of QPM genotypes.

Tabla 2. Desarrollo de genotipos QPM.

Name Pedigree/Background Gene(s) introgressed Country Reference

NB-Nutrinta (OPV) Poza Rica 8763 o2 Nicaragua Cordova (2001)

HQ INTA-993 (hybrid)

HB-PROTICTA (hybrid)

HQ-61 (hybrid)

HQ-31 (hybrid)

ICA- (hybrid)

FONAIAP (hybrid)

(CML144 × CML159) CML176

Nicaragua

Guatemala

El Salvador

Honduras

Colombia

Venezuela

Cordova (2001)

BR-473, BR-451 (OPV) o2 Brazil Cordova (2001)

INIA- (hybrid) CML161 × CML165 o2 Peru Cordova (2001)

HQ-2000 (hybrid) CML161 × CML165 o2 Vietnam Cordova (2001)

Zhongdan 9409 (hybrid) Pool 33 × Temp QPM o2 China Cordova (2001)

QUIAN2609 (hybrid) Tai 19/02 × CML171 o2 China Cordova (2001)

Susuma (OPV) Across 8363SR Mozambique, Senegal Krivanek et al. (2007)

Longe-5 ‘Nalongo’ (OPV) Across 8363SR Uganda Krivanek et al. (2007)

Obatanpa (OPV) Across 8363SR

Benin, Burkina Faso, Cameroon,

Cote d’Ivoire, Ghana, Guinea, Ma-

lawi, Mali, Nigeria, Senegal, South

Africa, Togo

Krivanek et al. (2007)

Lishe-K1(OPV) Across 8363SR Tanzania Krivanek et al. (2007)

EV 99 QPM (OPV) Cote d’Ivoire, Nigeria, Senegal,

Togo Krivanek et al. (2007)

KH500Q (hybrid) (CML144 × CML159) CML181 o2 Kenya Krivanek et al. (2007)

BHQP542 (hybrid) (CML144 × CML159) CML176 o2 Ethiopia Krivanek et al. (2007)

MHQ138 (hybrid) (CML144 × CML159) Pool15Q o2 Ethiopia Jilo (2022)

BHQPY545 (hybrid) CML181 × CML165 o2 Ethiopia Jilo (2022)

QS-7705 (hybrid) o2 South Africa Krivanek et al. (2007)

GH-132-28 (hybrid) P62, P63 o2 Ghana Krivanek et al. (2007)

ZS261Q (hybrid) (CZL01006 × CML176) ×

(CZL01005 × CML181) o2 Zimbabwe Krivanek et al. (2007)

441C (hybrid) CML142 × CML116 o2 Mexico Cordova (2001)

H-551C (hybrid) CML142 × CML150 o2 Mexico Cordova (2001)

H-553C (hybrid) (CML142 × CML150) CML176 o2 Mexico Cordova (2001)

H-519C (hybrid) (CML144 × CML159) CML170 o2 Mexico Cordova (2001)

H-368EC (hybrid) CML186 × CML149 o2 Mexico Cordova (2001)

H-369EC (hybrid) CML176 × CML186 o2 Mexico Cordova (2001)

V-537C (OPV)

V-538C (OPV)

Poza Rica 8763

Across 8762 o2 Mexico Gómez-M et al. (2003)

H-374C (hybrid) (CML176 × CML142) CML186 o2 Mexico Noriega González et al.

(2011)

H-564C (hybrid) (LT158 × LT159) LT160 o2 Mexico Sierra Macías et al. (2011)

V556AC (OPV) o2 Mexico Twumasi-Afriyie et al.

(2016)

ZAPATA 3 o2 Mexico Twumasi-Afriyie et al.

(2016)

ZAPATA 9 o2 Mexico Twumasi-Afriyie et al.

(2016)

V25 QPM (line) V25 × CML176 o2 India Babu et al. (2005)

Vivek QPM-9 (hybrid) VQL1 (CM212 × CML180) × VQL2

(CM145 × CML170) o2 India Gupta et al. (2009); Gupta et

al. (2013)

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Name Pedigree/Background Gene(s) introgressed Country Reference

BC2F4-1 (line) QCL3024 (o16) × QCL5019 (wx)

and QCL5008 (wx)o16 China Yang et al. (2013)

BQPM9 (line)

BQPM10 (line)

BQPM11 (line)

BQPM12, BQPM16 (line)

BQPM13, BQPM14 (line)

BQPM15 (line)

BQPM17 (line)

(B99 × CLQ 06901) B99

(B99 × CLRQ 00502) B99

(B100 × CLQ 06901) B100

(CLQ 06901 × B98) B98

(CLQ 06901 × B97) B97

(B91 × CLQ 06901) B91

(CLQ 06901 × B113) B113

o2 USA Worral et al. (2015)

ZPL 3 QPM (line)

ZPL 5 QPM (line)

ZPL 3 × CML144

ZPL5 × CML144 o2 Serbia Kostadinovic et al. (2016)

Zhao OP-6/o2o2 (line)Zhao OP-6 × QPM CA339

(pool33) o2 China Zhou et al. (2016)

BML-7 QPM (line) BML-7 × CML-186 o2 India Krishna et al. (2017)

CBML6 QPM (line)

CBML7 QPM (line)

DHM117 (hybrid)

BML6 × CML181

BML7 × CML181

CBML6 × CBML7

o2 India Surender et al. (2017)

HM4 QPM (line)

HM8 QPM (line)

HM9 QPM (line)

HKI323 × HKI161

HKI1105 × CMLI61

HKI1128 × HKI193-1

o2 India Hossain et al. (2018)

V238AC (OPV) Comiteco race (yellow) × CML-

172 o2 Mexico Coutiño Estrada and Váz-

quez Carrillo (2018)

Quality Protein Popcorn (QPP)

(lines)

CML154Q × (P2, P3, P9)

Tx807 × P2

K0326Y × (P3, P7)

o2 USA Ren et al. (2018)

QCL8006-1 (line) QCL8006-2

(line)

QCL3024 (o16) × Taixi19 (o2) and

QCL5019 (wx)o2/o16 China Wang et al. (2019)

HM5-A (hybrid)

HM12-B (hybrid)

(HKI1344 × PMI-102-o2o16) ×

(HKI1348-6-2 × PMI-102-o2o16)

(HKI1344 × PMI-102-o2o16) ×

HKI1378 × PMI-102-o2o16)

o2/o16 India Chand et al. (2022)

V56AC (OPV) Oloton race (yellow) × CML-172 o2 Mexico Coutiño Estrada et al.

(2022)

OPV: Open pollinated variety.

These results are consistent with a model in which the two

loci associated with mo2 genes inuence the expression of

γ-zein genes through dierent mechanisms: one aects the

transcription of the γ-zein locus and the other inuences the

stability of the γ-zein RNA. Holding et al. (2011) evaluated the

expression in developing endosperm (18 days after pollina-

tion, DAP) of QPM lines contrasting in vitreousness, reporting

a higher expression of the 27 kDa γ-zein gene and greater

accumulation of the protein in vitreous QPM lines compared

to o2 lines (Figure 1A). Wu et al. (2010) used RNAi to block

the expression of 27 kDa γ-zein in the CM105mo2 maize

line; and found that RNAi caused the reversal of the vitreous

phenotype to o2, demonstrating that 27 kDa γ-zein plays an

essential role in the modication of the endosperm in QPM.

Similar ndings were reported by Yuan et al. (2014) who used

γ-radiation mutagenesis to identify genes related to the

modication of the o2 mutant; they observed a generalized

decrease in α-zeins and an increase in γ-zeins (27 and 50 kDa)

in the K0326Y-QPM line compared to W64A+. The authors

proposed that the 27 kDa γ-zein plays an important role in

the formation of protein bodies and that the 50 kDa γ-zein,

despite being in a smaller proportion, could also be involved

in endosperm modication.

A genetic analysis in QPM RILs identied a locus (qγ27)

on chromosome 7 that results from the duplication of the 27

kDa γ-zein gene and causes an increase in gene expression

and the synthesis of 27 kDa γ-zein in QPM and wild-type

lines, conrming that the improved expression of 27 kDa

γ-zein is critical for endosperm modication in QPM (Liu et

al., 2016). The higher expression of 27 kDa γ-zein causes that

QPM endosperm accumulates a greater amount of small

protein bodies, which are suggested to allow the formation

of a more rigid vitreous matrix that resembles a wild type of

maize endosperm (Figure 3A) (Wu et al., 2010).

Alteration in starch composition and structure

The non-zein fraction in maize endosperm includes meta-

bolic enzymes that may also play a role in QPM endosperm

modication. Gibbon et al. (2003) performed a gel-based pro-

teomic analysis of non-zeins in maize near isogenic lines con-

trasting in vitreousness (CM105+, CM105o2 and CM105mo2),

and found that the vitreous QPM line showed an increased

accumulation of the enzyme granule-bound starch synthase

I (GBSSI), which is responsible for the synthesis of amylose.

The authors also found that amylopectin in the vitreous

endosperms showed a higher proportion of short branches

Volume XXV, Issue 2

Gonzalez-Nuñez et al: Mechanisms associated with endosperm modication / XXV (2): 79-89 (2023)

compared to that of normal and o2. These alterations in

starch structure may increase the proportion of amorphous

regions at the surface of starch granules, which favors their

compaction and the vitreous phenotype (Figure 3A). These

results suggested that starch biosynthetic enzymes may play

an important role in endosperm modication.

Genetic analyses of the cross between K0326Y-QPM

and W64Ao2 found a locus for vitreousness on chromosome

9 near genes involved in starch biosynthesis (Holding et al.,

2008; Holding et al., 2011). The biochemical characterization

of this locus, using RILs derived from the same cross, showed

that starch from vitreous mature endosperms had higher

levels of amylose and lower crystallinity compared to starch

from opaque endosperms, which was associated with lower

gelatinization enthalpy (Salazar-Salas et al., 2014). This beha-

vior was also observed by Juárez-García et al. (2013) who re-

ported lower enthalpy values in starches from vitreous lines

compared to those from opaque lines at the mature state.

These results could be explained by the higher proportion

of amylose and short branches of amylopectin in starch from

the vitreous lines. Short branches of amylopectin can reduce

crystal formation, while a higher proportion of long chains

can form more organized crystals that require higher tempe-

rature and gelatinization enthalpy (Jane et al., 1999). These

studies suggest that alterations in the amylopectin structure

play an important role in the modication of the endosperm.

Wu et al. (2015) reported that SSSIII may aect pullu-

lanase activity and indirectly inuence the vitreousness

of the kernel by altering the distribution and length of the

amylopectin glucan chains. Soluble starch synthase I (SSSI)

produces short chains with degrees of polymerization (GP)

of 8 - 12, while SSSII and SSSIII isoforms seem to be involved

in the formation of intermediate (GP 13 - 25) and long (GP

> 30) chains, respectively (Nakamura et al., 2005). A higher

proportion of amylopectin intermediate chains (GP 10 - 24)

and a decrease in the proportion of chains with GP of 25 - 40

was observed in starch from K0326Y-QPM and vitreous RILs

with respect to starch from W64Ao2 and opaque RILs, which

was associated with a higher proportion of amorphous

regions in the starch granules that favors their compaction

adopting polygonal shapes (Gibbon et al., 2003; Salazar-Salas

et al., 2014). This provides a mechanism that complements

the one associated with an increase in small protein bodies

rich in γ-zein (27 kDa) that ll the spaces between the starch

granules creating the vitreous phenotype (Figure 3A).

Genetic mapping of starch physicochemical properties

in RIL derived from K0326Y QPM and W64Ao2 identied

three loci on bins 4.05, 5.04, and 9.03 close to the starch

biosynthesis genes Brittle-2 (Bt2), Amylose extender-1 (Ae1),

and Waxy-1 (Wx1), respectively (Vega-Alvarez et al., 2022);

the analysis of gene expression in developing endosperm (30

days after pollination, DAP) showed that the transcript levels

of Wx1 were signicantly higher in K0326Y QPM and vitreous

RILs compared with W64Ao2 and opaque lines, which corres-

ponded to a greater GBSSI and amylose accumulation in the

vitreous lines at the same developmental stage. These results

are in agreement with those reported in mature endosperm

(Salazar-Salas et al., 2014) and conrms an important role for

GBSSI in the modication of the QPM endosperm. Jia et al.

(2013) analyzed the expression in developing endosperm

(22 DAP) of W64Ao2 and its normal counterpart and found

a lower expression of Wx1 in the opaque mutant. This study

also revealed that the expression of genes encoding pullula-

nase (Zpu1) and starch branching IIb (SBEllb) enzymes was

higher in W64Ao2 than W64A+. The regulation of these genes

may change the proportions of amylose and the branching

patterns of amylopectin in the starch granules of the o2

mutant contributing to the soft endosperm. Gonzalez-Nuñez

(2022) analyzed the activity of GBSSI and SBEIIb in develo-

ping endosperms (28 DAP) of K0326Y-QPM, W64Ao2 and RIL

contrasting in vitreousness; the GBSSI activity was higher

in the endosperm of the vitreous lines and was associated

with a higher proportion of amylose, whereas the activity of

SBEIIb was higher in opaque lines that showed higher levels

of amylopectin. These results support the hypothesis that en-

dosperm modication in QPM is associated with the synthe-

sis of starch with a higher proportion of amylose, which may

facilitate the packing of the starch granules resulting in the

vitreous phenotype (Figure 3A).

Modulation of carotenoid composition in amyloplast

envelope

Wang et al. (2020) identied Ven1 as a major QTL inuencing

the vitreous phenotype in the mature maize kernel. Ven1

encodes for the enzyme β-carotene hydroxylase 3, which

modulates the composition of carotenoids in the amyloplast

envelope. They observed that in the opaque endosperm is a

dysfunctional Ven1 allele that decreases the content of polar

carotenoids and increases that of non-polar carotenoids in

the amyloplast envelope, which provides greater stability

to this structure that under normal circumstances disap-

pears during kernel desiccation. The non-disruption of the

amyloplast envelope results in a poor interaction between

the protein bodies and the starch granules, leading to a soft

endosperm (Figure 3A).

Amelioration of the stress response by increasing the

energy availability

The development of the o2 endosperm involves a stress

response that reduces the energy levels and aects ATP-

dependent processes such as zein proteins synthesis (Li et

al., 2020). This may be due to a reduction in the expression

of pyruvate phosphate dikinase (PPDK1), aecting glycolysis

and the energy production. Holding et al. (2008) and Holding

et al. (2011) identied several dierentially upregulated ge-

nes in QPM, including pyrophosphate-dependent fructose-

6-phosphate 1-phosphotransferase (PFPα), a non-ATP-de-

pendent glycolytic enzyme. It was proposed that the higher

activity of PFPα in QPM compensates the reduced availability

of ATP in o2 endosperm (Guo et al., 2012). A cytosolic PPDK2

was also identied by these authors as an ATP-independent

glycolytic enzyme. Li et al. (2020) also found PFPα as a candi-

Volume XXV, Issue 2

Gonzalez-Nuñez et al: Biotecnia / XXV (2): 79-89 (2023)

date gene for endosperm modication in QPM and identied

cytosolic enolase (ENO) as another ATP-independent glyco-

lytic enzyme. Thus, the increased synthesis of enzymes that

do not require ATP for glycolysis in QPM provides energy by a

mechanism that is repressed in o2 endosperm.

CONCLUSIONS

There have been important advances in the understanding

of the mechanisms associated with endosperm modication

in QPM. However, the application of this information for the

ecient development of QPM materials is dicult due to the

multiple mechanisms involved in the creation of the vitreous

endosperm. So far, the increased accumulation of 27 kDa

γ-zeins seems to have the major contribution to the vitreous

phenotype, which is complemented with the alterations in

the composition and structure of the starch granules that

favor their compaction, as well as with alterations in the

composition of the amyloplast envelope that result in the

degradation of this structure during endosperm desiccation,

allowing a better interaction between protein bodies and

starch granules. These processes may not be possible without

the availability of energy provided by enzymes that enhance

the non-ATP-dependent glycolytic ux.

ACKNOWLEDGMENTS

This research was supported by grants from National Council

for Science and Technology CONACYT (167584 and 284552).

CONFLICTS OF INTEREST

The authors have no conict of interest to declare.

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PB PB

PB PB PB

PB PB

PBPB

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