African Crop Science Journal, Vol. 27, No. 4, pp. 653 - 677 ISSN 1021-9730/2019 $4.

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African Crop Science Journal by African Crop Science Society is licensed under
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DOI: https://dx.doi.org/10.4314/acsj.v27i4.8

PHYSIOLOGICAL AND BIOCHEMICAL BASIS FOR STAY-GREEN TRAIT

IN SORGHUM

I.K.A. GALYUON1,3, A. GAY1, A.K. BORRELL2 and C.J. HOWARTH1

Institute of Biological, Environmental & Rural Sciences, Aberystwyth University, Gogerddan,

1

Aberystwyth, SY23 3EE, UK

2
Queensland Alliance for Agriculture and Food Innovation (QAAFI), Hermitage Research Facility,
University of Queensland, Warwick, QLD 4370, Australia
3
Department of Molecular Biology & Biotechnology, University of Cape Coast, Cape Coast, Ghana
Corresponding author: [email protected]

(Received 5 February, 2019; accepted 19 November 2019)

ABSTRACT

Drought is a major cause of sorghum [Sorghum bicolor (L.) Moench] yield losses in rain-fed agriculture,
especially in the semi-arid and arid agro-ecological zones of Africa and Asia. Stay-green sorghum
genotypes are able to maintain grain filling under drought conditions. The trait has been employed in
the selection and breeding for post-flowering drought resistance, even though the genes regulating
the trait are still being identified. The objective of this study was to assess how leaf area and chlorophyll
are maintained in various sources of stay-green; and to determine whether the integrity of the
photosynthetic apparatus and enzymes involved in the maintenance of photosynthesis during post-
flowering drought stress are regulated differently. A glasshouse experiment was conducted using
three stay-green sorghum lines (B35, KS19 and E36-1) and a senescent control, R16, under well-
watered (WW) and water-limited (WL) conditions. The size of the canopy at anthesis varied
significantly between genotypes, and this profoundly impacted leaf senescence patterns. For example,
green leaf area (GLA) at anthesis was highly correlated with the decline in GLA during the first 21 days
of grain filling, under both WW (r = 0.92) and WL (r = 0.86) conditions. These differences in senescence
patterns were further exacerbated by the small pot size in this study (10 L). E36-1 is normally designated
as a stay-green genotype, but the growth of this ‘high leaf area’, genotype in a small pot resulted in a
senescent phenotype. Green leaf area retention was higher in B35 and KS19, and the loss of GLA
started 14 days earlier in the WL E36-1 and R16 plants, compared to B35, with little change in KS19.
Chlorophyll levels were higher in B35 and KS19 compared with R16 and E36-1 under WL conditions.
ΦPSII, CO2 assimilation rate, leaf conductance, transpiration rate and leaf water use efficiency were
higher in the stay-green genotypes under WL conditions compared to R16. Enzymes involved in leaf
nitrogen metabolism and chlorophyll biosynthesis, and photosynthesis were retained at higher levels
in the stay-green lines than in R16. Therefore, the stay-green mechanism resulted in reduced destruction
654 I.K.A. GALYUON et al.
of the photosynthetic apparatus, better nitrogen metabolism and chlorophyll turnover, and maintenance
of active enzymes involved in photosynthesis.

Key Words: Green leaf area, photosynthesis, Sorghum bicolor

RÉSUMÉ

La sécheresse est une cause majeure des pertes de rendement du sorgho [Sorghum bicolor (L.)
Moench] dans l’agriculture pluviale, en particulier dans les zones agro-écologiques semi-arides et
arides d’Afrique et d’Asie. Les génotypes de sorgho restant verts peuvent maintenir le remplissage
du grain dans des conditions de sécheresse. Le trait a été utilisé dans la sélection et la sélection pour
la résistance à la sécheresse après la floraison, même si les gènes régulant le trait sont encore en cours
d’identification. L’objectif de cette étude était d’évaluer comment la surface des feuilles et la chlorophylle
sont maintenues dans diverses sources de persistance; et pour déterminer si l’intégrité de l’appareil
photosynthétique et des enzymes impliqués dans le maintien de la photosynthèse pendant un stress
de sécheresse après la floraison est régulée différemment. Une expérience en serre a été réalisée en
utilisant trois lignées de sorgho vertes (B35, KS19 et E36-1) et un témoin de sénescence, R16, dans des
conditions bien arrosées (WW) et limitées en eau (WL). La taille de la canopée à l’anthèse variait
considérablement entre les génotypes, ce qui a eu un impact profond sur les profils de sénescence des
feuilles. Par exemple, la surface de la feuille verte (GLA) à l’anthèse était fortement corrélée au déclin de
la GLA au cours des 21 premiers jours de remplissage du grain, dans les conditions WW (r = 0,92) et
WL (r = 0,86). Ces différences de profils de sénescence ont été encore exacerbées par la petite taille du
pot dans cette étude (10 L). E36-1 est normalement désigné comme un génotype restant vert, mais la
croissance de ce «génotype de grande surface foliaire» dans un petit pot a entraîné un phénotype
sénescent. La rétention de la surface des feuilles vertes était plus élevée dans B35 et KS19 et la perte
de GLA avait commencé 14 jours plus tôt dans les plantes WL E36-1 et R16, par rapport à B35, avec peu
de changement dans KS19. Les niveaux de chlorophylle étaient plus élevés dans B35 et KS19 par
rapport à R16 et E36-1 dans des conditions de WL. FPSII, le taux d’assimilation du CO2, la conductance
des feuilles, le taux de transpiration et l’efficacité d’utilisation de l’eau des feuilles étaient plus élevés
chez les génotypes restant verts dans des conditions de WL par rapport à R16. Les enzymes impliquées
dans le métabolisme de l’azote des feuilles et la biosynthèse de la chlorophylle, ainsi que la
photosynthèse, ont été conservées à des niveaux plus élevés dans les lignes vertes restantes que
dans R16. Par conséquent, le mécanisme restant vert a entraîné une destruction réduite de l’appareil
photosynthétique, un meilleur métabolisme de l’azote et le renouvellement de la chlorophylle, ainsi
qu’un maintien des enzymes actives impliquées dans la photosynthèse.

Mots Clés: Zone de la feuille verte, photosynthèse, Sorgho bicolore

INTRODUCTION the crop is cultivated under rain-fed conditions,

often utilising stored soil moisture. Rainfall is
Sorghum [Sorghum bicolor (L.) Moench] is frequently erratic and patterns are changing
the fifth most important global cereal after due to climate change. Such conditions result
wheat, rice, maize and barley (FAO, 2014) for in crop losses.
food, feed, fibre and fuel; and is particularly Inadequate rainfall and soil moisture, during
well adapted to hot and dry conditions and after flowering in sorghum, can result in
(Paterson et al., 2009). A major constraint to serious yield losses due to premature plant
sorghum production is inadequate soil water death, stalk rot, lodging and reduced seed size
availability. In many parts of Asia and Africa, (Borrell et al., 2014a).
where sorghum is a staple to millions of people,
 Physiological and biochemical basis for stay-green trait in sorghum 655
Many drought resistant sorghum cultivars more accelerated leaf senescence in senescent
stay green until harvest, and the stay-green lines (Borrell and Hammer, 2000; Borrell et al.,
trait has been used for years by breeders as a 2001).
measure of post-flowering drought tolerance Genotypic differences in delayed onset and
(Borrell et al., 2000b; 2014; Jordan et al., reduced rate of leaf senescence were found
2012). The trait is characterised by the to be due to differences in specific leaf N and
retention of green stems and green upper N uptake during grain filling (Borrell and
leaves, even under severe post-flowering Hammer, 2000). Reduced CO2 assimilation
drought stress; and is associated with the caused by reduction in stomatal conductance,
maintenance of grain fill, reduced lodging, high reduced concentrations and activities of
stem carbohydrate content and resistance to photosynthetic enzymes, chlorophyll and N
charcoal stem rot under such conditions loss, among other factors, consequently limit
(Subudhi et al., 2000; Tao et al., 2000; the availability and partitioning of
Burgess et al., 2002; Borrell et al., 2000ab, photosynthates into grain filling. Grain yield
2014ab). Thus, delaying leaf senescence in sorghum under post-flowering drought
(stay-green) is an effective strategy for stress correlates positively with green leaf area
increasing cereal production under water- at mid-grain filling (Borrell et al., 1999) and
limited conditions (Mahalakshmi and Bidinger, green leaf area at maturity (Borrell et al.,
2002). 2000b), confirming that green leaf area
A number of different genetic sources of duration improves yields under drought stress.
stay-green have been identified and used in The longevity of leaves in stay-green sorghum
breeding programmes around the world might be promoted by a combination of several
(Borrell et al., 2000b, 2014ab; Jordan et al., biochemical factors, which interact to regulate
2012). It has been suggested that the origin of N remobilisation and chlorophyll turnover,
stay-green in sorghum is from perennial maintaining the integrity of the photosynthetic
landraces (Thomas et al., 2000), with reduced apparatus and enzyme activity as well;
monocarpic senescence compared with typical particularly, those involved in carbon and N
annual cereals. In some regions, such as East assimilation.
Africa, these perennial tendencies in locally The stay-green phenotype can be
adapted sorghums are exploited for ratooning categorised as “functional” for maintenance
to extend the productive period over two or of leaf photosynthesis or “non-functional”
more seasons (Escalada and Plucknett, 1975). (cosmetic), where chlorophyll degradation is
The longevity of leaves (defined as the time prevented but photosynthesis competence is
in days from initiation to when the leaf area is lost (Thomas and Howarth, 2000).
more than 90% yellow) is significantly reduced Senescence, as a whole, may be postponed or
for upper leaves at low nitrogen (Thomas and slowed down; alternatively, initiation may be
Rogers, 1990; Peng et al., 2013). Since the on schedule, but pigment metabolism may be
stay-green trait is characterised by delayed- compromised by a blockage in a biochemical
leaf-senescence, it can also be viewed as a pathway. Retention of chloroplast proteins,
consequence of a balance between supply and such as light-harvesting complex proteins II
demand for N during grain-filling (Borrell and (LHCPII), oxygen-evolving complex 33
Hammer, 2000; Thomas and Ougham, 2014). (OEC33) and Ribulose-1,5-bisphosphate
For example, if N supply from age-related carboxylase oxygenase (Rubisco), has been
senescence and N uptake during grain filling observed previously in sorghum containing the
are matched with grain N demand, the shortfall KS19 source of stay-green (de Villiers et al.,
in N supply for grain filling is greater in 1993), which explains, in part, how stay-green
senescent than stay-green hybrids, leading to sorghum could maintain photosynthesis for
656 I.K.A. GALYUON et al.
longer periods than their senescent KS19 is derived from a cross between
counterparts. However, these observations short Kaura, an important landrace cultivar
were made under non-drought conditions, from Nigeria, and Combine Kafir 60 (Henzell
whereas the trait is expressed under severe et al., 1984). E36-1 is a stay-green, tall, high-
drought stress (Srinivas et al., 2008). To yielding breeding line of the guinea-caudatum
maintain photosynthesis, the photosynthetic hybrid race with Ethiopian origin (van
apparatus and enzymes involved in carbon Oosterom et al., 1996). R16 is a high-yielding
assimilation, chlorophyll turnover and N cultivar from Maharashtra, India, which is
assimilation should be actively maintained in adapted to the post-rainy season (grown on
green leaves. Since different genetic sources stored soil moisture), but has a very rapid rate
of stay-green may employ different of leaf senescence (van Oosterom et al.,
mechanisms and inheritance characteristics 1996).
(Borrell et al., 2000ab; Vadez et al., 2011;
Jordan et al., 2012), assessment of these Plant culture and treatment. Plants were
factors should enhance the understanding of grown individually in 24.5-cm diameter pots,
the functioning of the stay-green trait. each containing 10 L of a mixture of soil, peat,
The objective of this study was to assess grit and perlite in a ratio of 3:3:3:1, in a
how leaf area and chlorophyll are maintained glasshouse at IBERS, Plas Gogerddan,
in various sources of stay-green; and to Aberystwyth, Wales, UK. Glasshouse
determine whether the integrity of the conditions were 30 °C in the day and 18 °C at
photosynthetic apparatus and enzymes night; and natural daylight was supplemented
involved in the maintenance of photosynthesis using 400 W high-pressure sodium lamps
during post-flowering drought stress are (Phillips so T Agro) for 12 hr daily. All plants
regulated differently. were watered to field capacity, until flowering.
Pots were arranged in a randomised block
MATERIALS AND METHODS design with 8 replicates. Blocking was intended
to address variation in the environment
Seed material. Three stay-green sorghum because the room used in the glasshouse was
genotypes (B35, E36-1 and KS19) were bordered to the East and West by other rooms
studied in comparison with a well-known with plants (Miscanthus sp) growing in them.
senescent sorghum genotype (R16). Sorghum These plants were tall and thus shaded the
seeds were obtained from the International eastern side in the early morning and the
Crops Research Institute for the Semi-Arid western side in the evening. Hence, there was
Tropics (ICRISAT), India, and the Queensland the need to block out the effect of shading in
Department of Agriculture and Fisheries’ the morning and evening.
Hermitage Research Facility, Australia. B35 At flowering (when anthers were visible
(also known PI534133 or SC35-6 or BTx642) on 50% of main shoot panicles), 4 replicates
is a BC1 selection from a converted (dwarf of each genotype remained well-watered
height, early flowering) version of IS12555 (WW) and 4 replicates were subjected to
durra sorghum, an Ethiopian landrace drought stress by limiting the amount of water
(Rosenow et al., 1983). It is a stay-green B- supplied. The water-limited (WL) plants were
line released as BTx642 (Rosenow et al., 2002) supplied with 250 ml water daily and the soil
and is the parent used in several QTL mapping moisture for the well-watered plants was
studies of drought tolerance in sorghum maintained at field capacity at all times.
(Walulu et al., 1994; Crasta et al., 1999; Xu All plants were supplied with fertiliser (NPK
et al., 2000). : 15-30-15) at 14 and 28 days after emergence
 Physiological and biochemical basis for stay-green trait in sorghum 657
and at swelling (growth stage 5 in sorghum). Leaf photosynthesis. Leaf gas exchange was
Fertiliser application was at a rate of 2 g pot-1 measured on a 5.6cm2 area of FL-3 using an
each time. Hence, 6 g of N per pot during the open portable gas exchange system (CIRAS-
experimental period. This was equivalent to 1, PP Systems, Hitchin Herts, UK). Carbon
300 kg of N ha-1 (Borrell et al., 2000a). dioxide (CO 2) assimilation rates (A), leaf
conductance (gL), transpiration rates (E) and
Measurement of green leaf area. Green intercellular CO 2 concentration data were
leaf area (GLA) was measured non- collected by placing each leaf in the cuvette
destructively on all leaves of four plants per maintained at 30ºC, with an ambient CO2
cultivar per treatment, at anthesis, using a concentration of 350 µL L-1 and exposed to
Delta-T Area Meter; and subsequently at photosynthetically active radiation (PAR) of
weekly intervals by calculation using the length 1100 µmol m-2 s-1. Measurements were taken
(from collar to tip) and maximal width of the on four plants per cultivar per treatment,
leaf. Preliminary studies using 180 leaves between 08:00 and 13:00 hr. Photosynthetic
indicated that the relationship between the water use efficiency (WUEL) was computed
product of the length and width and the size as the ratio of A/E.
measured using the area meter was: Chlorophyll fluorescence was measured in
the same leaves, used for gas exchange. An
y = 0.95 + 0.75x with an R 2 = 94.7% EARS Plant Photosynthetic Measurement
(P<0.001), (PPM) System was used to measure the
quantum yield of photosynthetic electron
Where: transport of photosystem II (Φ PSII ), as
described by Maxwell and Johnson (2000) in
y is the area measured using the area meter, light-adapted leaves. Three readings were
and x the product of the measured length and taken between the collar and tip of the leaf at
width for each leaf. equal spacing and averaged.
This relationship was then used to
determine the area according to the method Leaf proteins. Protein analysis was carried
described by Wolfe et al. (1988). The area of out at 0, 21 and 42 days after flowering (DAA).
each leaf was corrected for senescence by Protein was extracted from leaf sections cut
subtracting the area of the lamina lost to from the middle of the lamina of each leaf,
senescence defined by visible yellowing. avoiding the midrib as described by Smart et
al. (1995). Proteins were separated by SDS
Leaf chlorophyll levels. Non-destructive gel electrophoresis (Laemmli, 1970), using an
measurements of chlorophyll content were equal equivalent fresh weight loading per lane;
made at weekly intervals, using a SPAD-502 followed by Western blotting, using the method
chlorophyll meter (Minolta, Japan). SPAD described in Thomas et al. (1999). Individual
values for sorghum have been found to be polypeptides were visualised using a
highly correlated with total leaf chlorophyll, chemiluminescence detection kit (Roche),
determined by spectrophotometry (Xu et al., according to the protocol of the
2000). Nine readings were taken along each manufacturers, using antibodies (Hilditch et
side of the lamina of the fourth leaf from top al., 1989; Davies et al., 1990; Smart et al.,
(flag leaf minus 3; FL-3) in four plants per 1995). Bands were quantified following
treatment from 14 to 49 days after flowering background correction, using ImageJ
(DAA) and the average calculated. (Rasband, W.S., ImageJ, U. S. National
658 I.K.A. GALYUON et al.
Institutes of Health, Bethesda, Maryland, USA, RESULTS
http://imagej.nih.gov/ij/, 1997-2016).
Green leaf area (GLA) and chlorophyll
Grain yield. Panicle yield was determined retention. Loss of chlorophyll and a
after oven drying at 60 ºC for 48 hours, after consequent decline in green leaf area are the
harvest. Panicles were hand-threshed and seed visible expression of leaf senescence. The
weight per panicle, and 100 seed weight genotypes studied here varied considerably in
measured. The 100 seed weight and total seed their retention of green leaf area (GLA) from
weight per panicle were used to determine the flowering to physiological maturity, under both
number of seeds per head as described by well-watered (WW) and water-limited (WL)
Borrell et al. (2000a), viz; conditions (Table 1). Total green leaf area
(GLA) per plant at anthesis was highest in E36-
Number of seeds per panicle (g) = 1, intermediate in B35 and R16, and lowest in
KS19 (Fig.1). Loss of GLA was rapid in E36-
Panicle seed weight x 100 (g) 1 and R16 plants under both WW and WL
Weight of 100 seeds conditions between 0 and 28 days after
anthesis (DAA). Indeed, by 21 DAA, E36-1
Harvest index (HI) and the ratio of grain and R16 had lost 72% of GLA under water
yield to panicle yield (panicle harvest index limitation; while the reductions under WW
(PHI)) for each plant were determined conditions were 54 and 39% for E36-1 and
(Bidinger et al., 1987). R16, respectively (Fig. 1).
In B35, the decline in GLA was not
Statistical analysis. Data were analysed using apparent until 14 DAA; and the rate of loss of
Minitab Release 13 software. Significance of GLA was slow thereafter (Fig. 1). Differences
differences between treatment means was between the WW and WL plants began to show
determined by using ANOVA and LSD from 21 DAA onwards, with reductions of 16
(Fisher’s). and 29%, respectively, for the WL and WW

TABLE 1. Sorghum green leaf area (cm2) at various days after flowering in well-watered (WW) and
water-limited (WL) plants

Line Treatment Days after flowering

0 21 42

B35 WW 4635 3908 2977

WL 4635 3299 2206
E36-1 WW 7003 3238 1477
WL 7003 1994 921
KS19 WW 1881 1796 1282
WL 1881 1758 1005
R16 WW 4884 3000 1286
WL 4884 1392 988

LSD0.05 86.8 77.6 75.8

 Physiological and biochemical basis for stay-green trait in sorghum 659
8000

7000

6000
Green leaf area per plant (cm2)

5000

4000

3000

2000

1000

0
0 7 14 21 28 35 42 49
Days after flowering

Figure 1. Changes in sorghum green leaf area in sorghum plants. Plants were either well watered
(WW) and water-limited (WL) from flowering to physiological maturity. Values are means of four
replicates.

plants. In contrast, the loss of GLA in KS19 senescence. Age-related senescence continued
was slow in both the WL and WW plants, and throughout the experiment, with R16 senescing
by 21 DAA they had lost only 7 and 5%, at twice the rate (1.75% loss in GLA per day)
respectively. The loss of GLA increased in than B35 (0.85% loss in GLA per day). KS19,
KS19 plants, thereafter, and by 42 DAA it had which had the smallest canopy size at anthesis,
reduced by 32% (WW) and 47% (WL) did not display senescence until 28 DAA,
compared to that at flowering (Table 1). The whereas E36-1, with the largest canopy size
loss of GLA was generally more rapid in plants at anthesis, was exhibiting significant
with high GLA at anthesis, such as E36-1 (Fig. senescence by 7 DAA.
2). Nonetheless, B35, which had similar size
of GLA as R16, had a slower rate of Stress-induced senescence (WL
senescence than R16. treatment). The decline in green leaf area
(GLA) under water-limited conditions during
Age-related senescence (WW treatment). the first 21 days after anthesis was positively
GLA at anthesis was positively correlated with correlated with the loss in GLA per day (0-21
percent loss in GLA per day (0-21 DAA; r = DAA; r = 0.86; Fig. 2b). But again, R16
0.92; Fig. 2a). However, R16 (senescent) and (senescent) and B35 (stay-green) exhibited
B35 (stay-green) exhibited similar canopy sizes similar canopy sizes at anthesis (~4700 cm2),
at anthesis (~4700 cm2); yet the decline in GLA yet the decline in GLA (Fig. 2) during the first
during the first 21 DAA was significantly 21 DAA under WL conditions was significantly
(P<0.05) higher in R16 (39%) than B35 (16%); higher in R16 (72%) than in B35 (29%),
indicating inherent differences in age-related indicating inherent differences in stress-
660 I.K.A. GALYUON et al.
A Well watered (WW)
Percentage loss GLA/day (0-21 DAA)

Green leaf area at anthesis (cm2 plant-1)

B Water-limited (WL)
Percentage loss GLA/day (0-21 DAA)

Green leaf area at anthesis (cm2 plant-1)

Figure 2. The relationship between sorghum green leaf area at anthesis (cm2 plant-1) and the percentage
loss in green leaf area (GLA) per day (between 0 and 21 DAA) for four sorghum genotypes grown
under (A) well-watered, and (B) water-limited conditions.

induced senescence. E36-1, which had the R16 plants; whereas in the WL B35 and KS19
largest canopy size at anthesis, senesced at plants, the decline occurred after 7 and 28
the same rate as R16 (3.4% per day). DAA, respectively. Once senescence had
The retention of GLA (%GLA) was greater started, KS19 senesced at a greater rate (2.0%
in the B35 (84%) and KS19 (98%) than in E36- per day) than B 35 (1.6% per day). There was
1 (62%) and R16 (46%) under WL, compared a significant difference in GLA between WL
to WW plants at 21 DAA. In all genotypes, and WW R16 plants at 14 DAA; whereas this
water limitation either brought forward the was not apparent between WL and WW B35
onset of leaf senescence and/or enhanced the plants until 35 DAA.
rate of leaf senescence. GLA declined rapidly Among the WW plants, chlorophyll levels
from flowering in the water-limited E36-1 and of FL-3 were highest in B35 and KS19
 Physiological and biochemical basis for stay-green trait in sorghum 661
throughout the sampling period, with no R16, particularly, in the second and third
significant differences between them or weeks after flowering. In B35 and KS19, the
between flowering and physiological maturity differences in chlorophyll levels between WW
(Fig. 3). R16 also had higher levels of and WL plants were not significant until 22
chlorophyll than E36-1 when well-watered, and 35 DAA, respectively.
but both genotypes displayed a steady reduction
in chlorophyll levels during the course of the Leaf photosynthesis and water use
experiment. efficiency. Quantum yield of PSII electron
Chlorophyll levels were reduced in all the transport (ΦPSII) declined slowly in the well-
WL plants compared to the WW plants, but watered plants (Fig. 4). There was no
the timing of the onset of this reduction differed significant difference between cultivars,
between genotypes. In E36-1 and R16, except during the last two weeks of sampling,
chlorophyll levels were significantly (P<0.05) when R16 and E36-1 plants had significantly
reduced by water limitation compared to the lower ΦPSII (Fig. 4). For B35 and KS19 plants,
WW plants, but the reduction was immediate there was no difference in ΦPSII between WW
in E36-1 and delayed by one week in R16. and WL treatments throughout the sampling
However, the reduction was more rapid in period, except at the last sampling date when

65

55

45
SPAD reading

35

25

15

0
1 8 15 22 29 36 43
Days after flowering
Figure 3. SPAD readings showing levels of chlorophyll retained in well-watered (WW) sorghum
plants and water-limited (WL) from 14 to 49 DAA. Values are means of 4 replicates.
662 I.K.A. GALYUON et al.

70

60
ΦPSII (%)

50

40

30
1 8 15 22 29 36 43
Days after flowering
Figure 4. Changes in quantum efficiency of photosystem II (ÖPSII) in well-watered (WW) and water-
limited (WL) sorghum plants from flowering to physiological maturity. Values are means of 4 replicates.

it was significantly (P<0.05) lower in WL for than in E36-1 and R16 at the end of week 3.
KS19 plants. Water limitation significantly At physiological maturity, under both WW and
(P<0.05) reduced Φ PSII in E36-1 and R16 WL treatments, A was significantly (P<0.05)
compared to the WW treatment from 15 to 43 higher in B35 and KS19 than in E36-1 and R16.
DAA, in parallel to the loss in chlorophyll (Fig. Leaf conductance was significantly
4). (P<0.05) reduced by water limitation in B35,
Carbon dioxide assimilation rate (A) and E36-1 and R16 at the end of the third week
leaf conductance (gL) were higher in R16 than (Fig. 5); whereas in KS19 there was no
any of the other lines at flowering; but declined difference between the WW and WL plants.
sharply thereafter in both the WW and WL However, the absolute values of gL were higher
plants (Fig. 5). By the end of the third week, in B35 and KS19 than in E36-1 and R16 under
A was reduced significantly (P<0.05) in E36- WL conditions. Hence, under WL conditions,
1 and R16 compared with the respective WW B35 and KS19 were able to reduce water
plants. The reductions were 68 and 59% in in losses via transpiration better than E36-1 and
R16 and E36-1, respectively, compared to only R16.
20 and 14% reductions in B35 and KS19, Leaf conductance correlated with CO 2
respectively. Furthermore, in the water-limited assimilation rate, with the coefficients of 0.77
treatment, carbon dioxide assimilation rate was (P<0.001), 0.73 (P<0.001), 0.52 (P <0.01) and
significantly (P<0.05) higher in B35 and KS19 0.74 (P<0.001) for B35, E36-1, KS19 and R16,
 Physiological and biochemical basis for stay-green trait in sorghum 663

Figure 5. Effect of water limitation on CO2 assimilation rate (A) and leaf conductance (gL) at the end
of 1, 3 and 6 weeks after sorghum flowering. Plants were either well-watered (WW) or water-limited
(WL). Means are values of 4 replicates. Vertical lines above are the LSD values at P = 0.05 for the
corresponding sampling date.
664 I.K.A. GALYUON et al.
respectively; under well-watered conditions. Transpiration rate (E) was also significantly
The corresponding coefficients for the water- (P<0.05) higher in R16, compared to the stay-
limited plants were 0.65 (P<0.001), 0.82 green lines at flowering (Fig. 6). Generally, E
(P<0.001), 0.82 (P<0.001) and 0.81 was lower in the WL plants and corresponded
(P<0.001), respectively, for B35, E36-1, KS with gL, indicating that stomatal conductance
19 and R16. mainly regulated transpiration. The R-square

Figure 6. Effect of water limitation on transpiration rate (E) and photosynthetic water use efficiency
(WUEL) at the end of 1, 3 and 6 weeks after sorghum flowering. Plants were either well-watered (WW)
or water-limited (WL). Means are values of 4 replicates. Vertical lines above are the LSD values at P =
0.05 for the corresponding sampling date.
 Physiological and biochemical basis for stay-green trait in sorghum 665
values were 90.9% for B35 and E36-1, 81.6% the well-watered (WW) and water-limited
for KS19 and 86.1% for R16, under well- (WL) plants in all lines (Table 3). However,
watered conditions. The corresponding values the values of these parameters were higher in
for B35, E36-1, KS19 and R16 water-limited all the stay-green lines than in the senescent
plants were 93.7, 95.5, 89.7 and 86.5%, cultivar. The harvest index (HI) in all three
respectively. For R16, transpiration rate was stay-green lines was significantly (P<0.05)
significantly (P<0.05) reduced at the end of higher than in R16. B35 and E36-1 also had
the third week in WL plants (by 72%), significantly (P<0.05) higher number of seeds
compared with WW plants (by 44%). Similarly, and grain dry mass than R16. Grain dry mass
in E36-1, E was significantly (P<0.05) was not reduced in KS19, with minimal
reduced (67%) by water limitation, compared reductions in E36-1 (8%) and B35 (13%) due
to the WW (28%) from flowering at the end to water limitation, compared to the significant
of the third week. For B35 and KS19, water (P < 0.05) reduction of 35% in R16. However,
limitation did not have a significant (P>0.05) grain dry mass was still low in KS19 under
effect on E at any sampling dates. WW conditions, similar to R16.
Leaf water use efficiency (WUE L ), Panicle harvest index (PHI) was also higher
computed as A/E, generally declined from in the stay-green lines than in R16, where it
flowering and the effect of water limitation was significantly (P<0.05) reduced in WL
was most apparent at the end of the third week compared to WW. PHI did not differ between
from flowering (Fig. 6). In B35, there was B35 and E36-1, but significantly (P<0.05)
no difference between the WW and WL lower in KS19. These results indicate that
treatments; while in KS19, E36-1 and R16, water deficit reduced seed set in B35 and R16
WUE L was reduced by 11, 28 and 39%, by 13 and 21 %, respectively; and reduced
respectively, by water limitation at the end of seed size in E36-1 and R16 by 6 and 18 %,
the third week; indicating a genotype by respectively.
treatment interaction. However, water
treatment had no impact on WUEL at week 6. DISCUSSION
These findings indicate that WUEL was greater
in the stay-green lines under WL conditions. Findings from the present study indicate that
stay-greenness in the three stay-green
Specific proteins. Trends in band intensity sorghum genotypes may have different
of a range of photosynthetic, nitrogen physiological and biochemical basis and can
metabolism and other leaf proteins assessed be influenced by the pot size for glasshouse
at 21 DAA and at physiological maturity (42 studies. All WL plants were given the same
DAA) in relation to that at flowering are limited amount of water per day, and pot size
presented in Table 2. In both B35 and KS19, was constant. The E36-1 plants had much
these proteins were not affected by water greater leaf area per plant at anthesis (Fig. 1),
limitation. For E36-1, only D1 proteins, thus would use more water, and would be more
OEC33, GS1 and Glu-t-RNAL were reduced stressed. Hence, in these plants, WL induced
in the water-limited, compared to the well- rapid senescence which allowed limited
watered plants. For the senescent R16 plants, expression of stay-green. However, the yields
D1, cytochrome f, LHCPII, PEPCK, PEPC, under WL had some degree of protection as
Glu-t-RNAL and GSAAT were reduced by there was only non-significant difference in
water limitation. yield (62.5 SEM 7.9 and 57.2 SEM 3.7 g
plant-1 in WW and WL, respectively). By a
Grain yield. Seed number per plant, grain similar argument, the smallest plants at
dry mass per plant (mean seed weight) and HI anthesis (KS19) used the least amount of water
did not differ significantly (P>0.05) between and were not very stressed, and had similar
666 I.K.A. GALYUON et al.
TABLE 2. Band intensity (%) relative to that at sorghum flowering time. Plants were either well-
watered (WW) or water-limited (WL). Band were quantified following background correction using
ImageJ

21 DAA 42 DAA

WW WL WW WL

D1 protein
B35 89.3 88.7 91.7 86.9
KS19 103.1 94.4 97.4 94.2
E36-1 94.9 66.1 68.4 59.7
R16 99.3 82.6 55.8 44.0

OEC33
B35 100.1 98.8 100.3 97.2
KS19 99.2 97.7 102.6 99.3
E36-1 107.6 96.9 95.8 56.8
R16 85.5 79.5 66.7 65.5

Cytochrome f
B35 105.6 97.5 98.6 100.3
KS19 106.0 97.4 103.8 97.3
E36-1 66.9 63.2 22.8 25.2
R16 105.1 71.8 48.9 30.7

LHCP11
B35 100.0 100.6 100.4 111.0
KS19 110.6 134.1 103.8 127.2
E36-1 100.3 78.5 36.8 34.6
R16 96.9 75.9 82.6 35.7

PEPCK
B35 112.5 90.1 29.7 31.7
KS19 145.3 156.4 102.3 98.3
E36-1 36.6 17.0 6.7 6.4
R16 145.6 103.0 88.6 51.2

PEPC
B35 116.1 100.7 103.4 105.6
KS19 92.3 93.1 82.0 79.3
E36-1 84.2 80.9 40.0 43.6
R16 114.9 107.0 72.8 28.9

Rubisco (large sub-unit)

B35 102.1 101.0 104.5 101.4
KS19 101.2 101.1 99.2 101.9
E36-1 101.5 104.4 103.4 99.1
R16 101.2 99.1 98.2 103.3
 Physiological and biochemical basis for stay-green trait in sorghum 667
TABLE 2. Contd.

21 DAA 42 DAA

WW WL WW WL

GS1
B35 101.8 102.2 101.8 104.9
KS19 103.7 103.7 101.9 102.2
E36-1 419.2 277.1 499.4 277.0
R16 113.6 167.9 206.6 187.8
GS2
B35 99.2 101.8 104.4 103.7
KS19 104.7 103.7 99.5 101.4
E36-1 98.0 93.0 90.5 92.8
R16 88.6 90.6 83.1 76.5

Glu-t-RNAL
B35 104.9 98.4 103.7 104.5
KS19 101.6 104.5 103.7 102.8
E36-1 80.6 61.0 73.5 78.2
R16 92.0 103.6 93.7 73.7

GSAAT
B35 91.3 97.1 117.9 116.1
KS19 101.6 99.5 102.2 102.1
E36-1 101.1 83.9 92.8 84.2
R16 98.9 119.0 102.7 86.5

PORB
B35 69.7 55.3 53.1 44.4
KS19 108.0 131.5 90.3 92.7
E36-1 n.d. n.d. n.d. n.d.
R16 104.6 53.9 26.1 27.9

D1 protein = the reaction centre polypeptide of photosystem II; OEC33 = oxygen evolving complex;
GS1 = cytoplasmic glutamine synthetase; Glu-t-RNAL = Glutamyl-tRNA ligase: EC 6.1.1.17; LHCPII =
light-harvesting complex proteins II; PEPCK = phosphoenolpyruvate carboxykinase; PEPC =
phosphoenolpyruvate carboxylase; GSAAT = glutamic semi-aldehyde aminotransferase

yields in WW and WL. By comparison, the g plant-1 in WW and WL respectively) whilst

stay green had a much higher leaf area at the there was a much larger proportional reduction
end of the experiment under WL (2206 and in yield by WL in the genotype without stay
988 cm2 for B35 and R16, respectively), than green, R16 (yield 10.2 SEM 0.8 and 6.6 SEM
the two genotypes (B35, stay-green, and R16 0.7 g plant-1 in WW and WL respectively. Thus
not stay green) with intermediate leaf area at overall this reinforces the value of the stay
anthesis. Furthermore, there was only a green character in maintaining yield under
slight reduction in yield by WL in the stay green water limitation and shows it has no negative
B35 (yield 44.9 SEM 2.8 and 38.9 SEM 4.1 impact under lower stress levels.
668 I.K.A. GALYUON et al.

TABLE 3. Sorghum grain yield parameters of well-watered (WW) and water-limited (WL) plants

Line Treatment Seeds/plant Mean seed PHI (%) HI (%) Mean 100
weight (g) seed weight (g)

B35 WW 1185.5 ± 69.7 44.91 ±2.82 84.96 ± 0.41 36.36 ± 1.54 3.79 ± 0.11
WL 1027.0 ±119.9 38.92 ± 4.13 83.87 ± 1.73 33.33 ± 3.02 3.81 ± 0.06
E36-1 WW 1593.0 ±231.0 62.45 ± 7.93 85.98 ± 0.50 23.44 ± 2.13 3.95 ± 0.09
WL 1543.0 ± 98.4 57.21 ± 3.69 85.90 ± 0.11 25.15 ± 1.43 3.71 ± 0.03
KS19 WW 421.1 ± 96.2 10.06 ± 2.42 75.07 ± 2.65 26.59 ± 3.66 2.38 ± 0.06
WL 447.0 ±115.0 10.32 ± 2.38 72.98 ± 2.71 26.29 ± 3.42 2.38 ± 0.11
R16 WW 269.9 ± 22.5 10.21 ± 0.82 50.31 ± 1.36 7.52 ± 0.73 3.79 ± 0.10
WL 214.4 ± 26.5 6.59 ± 0.74 40.33 ± 3.05 5.56 ± 0.66 3.09 ± 0.08
LSD0.05 335.22 11.04 5.54 6.85 0.24

Values are Means ± SEM of 4 replicates; PHI = Panicle harvest index, HI = Harvest index

Biochemical data relating to leaf senescence factors could have impacted on senescence
can only be interpreted with understanding of patterns and grain yield. Indeed, a reduction
whole plant physiology. Hence, three key in N uptake and assimilation has been reported
limitations to the current study, likely to in previous studies on different plant species
confounded leaf senescence patterns include: with restricted soil volume for root growth
(i) the pot size was small, (ii) there were large (Ronchi et al., 2006; Zhu et al., 2006; Yang et
genotypic variations in leaf size at anthesis, al., 2007). Hence, the results of our study need
and (iii) the four genotypes evaluated were not to be considered in the context of this
from a common genetic background. limitation.
In an experiment to determine the impact
of pot size on root and shoot growth (Yang et Canopy size at anthesis. The size of the
al., 2010), small pots (less than 28 litres in canopy at anthesis varied greatly between
volume) significantly inhibited tillering and genotypes, and this profoundly impacted leaf
shoot growth for both sorghum and maize senescence patterns (Fig. 1). For example at
plants. Although the same total amount of slow anthesis, the GLA of E36-1 (7003 cm2) was
release fertiliser was applied to large and small almost four times that of KS19 (1881 cm2)
pots during pot filling in the study of Yang et and since the WL plants were supplied with
al. (2010), visible signs of nitrogen deficiency only 250 ml water daily after anthesis,
were obvious in lower leaves of plants growing regardless of genotype, those genotypes with
in small pots. This suggests that the small pot a larger leaf area would have used more water
size in our experiment (10 L) affected tillering, than those with a smaller leaf area, thereby
shoot growth, root:shoot ratio and N uptake. potentially affecting leaf senescence patterns.
In our study, fertiliser was supplied at In fact, the percentage decline in leaf area
intervals during the study to augment the effect during the first 21 days after anthesis was in
of nitrogen deficiency that would have resulted the following order: E36-1 (72%), R16 (71%),
from limiting root expansion in the small- B35 (29%) and KS19 (7%).
volume growth pots. However, different This decline in leaf area highly correlated
nutrient requirements due to differences in plant (r = 0.87) with the initial canopy size at
sizes could have impacted senescence anthesis: E36-1 (7003 cm2), R16 (4884 cm2),
differently among the genotypes. All of these B35 (4635 cm 2 ) and KS19 (1881 cm 2).
 Physiological and biochemical basis for stay-green trait in sorghum 669
Therefore, to a large extent, senescence maturity and height (this is why near-isogenic
patterns were determined by the initial size of lines are commonly used in physiological
the canopy at anthesis; hence, leaf senescence studies to remove such confounding factors).
patterns need to be interpreted in light of canopy Nonetheless, the current findings confirm B35
size at anthesis. Indeed, Borrell et al. (2014a) and KS as stay-green genotypes with different
found that Stg loci reduce canopy size at physiological mechanisms for staying green
flowering by modifying tillering, leaf number after anthesis under drought stress.
and leaf size; and that smaller canopy size at
flowering reduces pre-anthesis water use, Green leaf area and chlorophyll retention.
which under post-flowering water stress The results presented here indicate that the
increases water availability during grain filling drought treatment used was sufficient to
and, consequently, grain yield. Thus, the prevent premature plant death and enable the
designation of E36-1, which has been trait to be expressed. Two types of senescence
identified as one of the best sources of stay- were assessed in this study: age-related (WW)
green (Badigannavar et al., 2018), was not and stress-induced (WL). Clearly, B35 and
confirmed in this study. KS19 demonstrated the stay-green trait by
Maintenance of photosynthesis during grain maintaining green canopy, even at maturity
filling is likely to be highly associated with GLA (Badigannavar et al., 2018), but through
at anthesis in this study, since this factor was different mechanisms. E36-1 behaved as the
the key driver of leaf senescence. Indeed, senescent genotype, R16, maybe due to the
photosynthesis is dependent on available green factors already discussed under the section for
leaf area, which can be reduced by senescence, small pot.
thus reducing canopy phosynthesis
(Badigannavar et al., 2018). Photosynthesis. Photosynthesis, measured
Changes in other parameters during grain as quantum yield of photosystem II and carbon
filling, including chlorophyll proteins, quantum dioxide assimilation (Fig. 2) indicated that the
yield, and CO2 assimilation, were largely a stay-green sorghum genotypes maintained
consequence of differences in GLA at anthesis. photosynthesis for longer periods compared
Therefore, biochemical data needs to be to the senescent R16. Stay-green sorghum
interpreted in relation to whole plant genotypes retain green leaf area during grain
physiology. filling for longer periods than go-browns
(Borrell et al., 2000ab, 2014; Harris et al.,
Genetic background. The four genotypes 2007), presumably maintaining photosynthetic
evaluated in this study (B35, KS19, E36-1 and capacity for longer in stay-green lines. The
R16) were selected to represent different retention of chloroplast proteins, such as
sources of stay-green. However, they differed LHCPII, OEC33 and Rubisco, until late in
significantly in other traits such as maturity senescence observed in the KS19 source of
and plant height. Understanding how genetic stay-green indicates that photosynthesis may
and biochemical mechanisms contribute to be maintained for longer periods during
phenotypic responses of crop plants should senescence in these genotypes (de Villers et
provide opportunities for breeding and al., 1993).
selecting genotypes for various environmental Findings in our study have confirmed that
conditions (Boyles et al., 2019). Therefore, photosynthesis is maintained for longer periods
any comparisons in the physiological or in stay-greens under water limitation. Quantum
biochemical mechanisms underpinning yield (ΦPSII), a measure of quantum efficiency
drought adaptation in these lines, will likely be of CO 2 assimilation (number of quanta
confounded by variation in other traits e.g. absorbed per mole of CO2 reduced), is a good
670 I.K.A. GALYUON et al.
guide to photosynthetic functionality of leaves increased in B35 and KS19 plants. These
(Maxwell and Johnson, 2002). Φ PSII was findings confirm that B35 and KS19 plants
reduced by water limitation and declined 14 were drought tolerant since drought tolerance
days earlier in R16 compared to B35 and KS19. in sorghum is associated with the ability to
The comparison between R16 and B35 is maintain high stomatal conductance,
particularly insightful since the confounding transpiration rate and photosynthesis under
effect of leaf size at anthesis was largely drought stress (Tsuji et al., 2003).
removed.
B35 and KS19 plants, which had higher Chlorophyll biosynthetic pathway. The
ΦPSII, also had higher CO2 assimilation rates abundance of both Glu-tRNAL and GSAAT
(Fig. 5). The reduction CO2 assimilation rates proteins did not change much between
in B35 and KS19 by water limitation was not flowering and physiological maturity in any of
significant (P>0.05). Though the reduction in the stay-green genotypes studied under both
E36-1 was significant (P<0.05), the decline WW and WL conditions (Table 2). This
was also more gentle compared to R16. indicates that chlorophyll biosynthesis and
However, as explained earlier, E36-1 exhibited turnover were maintained in these genotypes,
a large GLA at anthesis, and combined with unlike in R16, in which both proteins were
the small pot size, resulted in leaf senescence. reduced by water-limitation at 42 DAA (Table
Nonetheless, our findings confirm that 2). Glutamate-tRNA ligase (Glu-tRNAL) and
photosynthesis is maintained for longer periods GSAAT are important in the biosynthesis of
in stay-green genotypes, when water is chlorophylls (Czarnecki and Grimm, 2012).
limiting. The maintenance of photosynthesis Therefore, the maintenance of these proteins
could also be explained, in part, as a result of for longer periods partially accounted for
chlorophyll retention, since chlorophyll is a prolonged photosynthesis in the stay-green
major chloroplast component for genotypes.
photosynthesis (Anjum et al., 2011). However, POR B is the constitutively expressed and
the retention of chlorophyll post-anthesis was the only remaining POR in light-grown plants
likely a consequence of GLA at anthesis. (Vavilin and Vermaas, 2002). It persisted in
Leaf conductance (gL) and transpiration KS19 and was not detectable in leaves of E36-
rates (E) were also higher in the stay-green 1 from flowering to physiological maturity
plants compared to the senescent R16 cultivar under both WW and WL conditions. The
(Fig. 5). Stomatal conductance is a measure higher amounts of POR B in B35 compared
of transpiration rate and photosynthetic with R16 at 42 DAA, despite equivalent GLAA,
potential of plants under drought stress supports the involvement of this enzyme in
(Badigannavar et al., 2018). In R16 plants, the the maintenance of chlorophyll turnover in
reduction in leaf conductance and transpiration stay-green genotypes, particularly under water
rates resulted in a corresponding reduction in limitation.
CO2 assimilation rate (A), which is in agreement
with reports by Anjum et al. (2011) that Glutamine synthetase (GS) isoenzymes.
reduction in CO2 assimilation is a consequence Abundance of GS isoenzymes in the cytosol
of reduced stomatal conductance by drought (GSI) and chloroplast (GSII) did not change
stress. B35, which had similar GLA as R16 at much from flowering to physiological maturity
anthesis, maintained photosynthesis for longer (Table 2). The exception was the reduction in
periods under both WW and WL conditions. GSII in R16. Glutamine synthetase (GS)
Furthermore, whereas transpirational water catalyses the synthesis of glutamine from
use efficiency (WUEL) declined with water ammonium and glutamate (Németh et al.,
limitation in R16 and E36-1, WUE L was 2018). These authors argue that GS plays a
 Physiological and biochemical basis for stay-green trait in sorghum 671
central role as a regulator between the nitrogen result of drought, therefore, would lead to a
and carbon cycles by maintaining the decline in CO2 assimilation since electron
glutamine-glutamate pool in the chloroplast on transport would be adversely affected. Since
the level of substrates, in addition to its the abundance of OEC33 did not change much
ammonia assimilation function. We found no between the well-watered and water-limited
differences in GSI proteins between the well- stay-green plants (B35 and KS19 (Table 2) it
watered and water-limited plants, except that could partially account for the higher and
GSI was reduced in E36-1 under water prolonged photosynthetic rates. R16 and E36-
limitation. 1 displayed reductions in OEC33 of 18 and
Re-assimilation of ammonium by GSII is 41%, respectively, under WL conditions.
crucial to plants as levels of ammonium The stay-greens (B35 and KS19)
released during photorespiration could be more maintained a greater proportion of D1 proteins
than the primary N assimilation (Igamberdiev between flowering and physiological maturity,
et al., 2014)). For instance, barley (Hordeum compared to the senescent R16 under water
vulgare) mutants defective in GSII and limitation (Table 2). Reactive oxygen species
containing low amounts of GSI grown in air, degrade D1 protein and inactivate PSII (Miyake
died because they were unable to re-assimilate et al., 2005). Damaged D1 protein is removed
ammonium released from photorespiration and degraded, and the PSII complex repaired
(Oliveira et al., 2002). Hence, the loss of by prompt insertion of newly synthesised D1
chlorophyll in R16 under water-limitation might protein (Henmi et al., 2003). This implies that
have resulted from reduction in the abundance the integrity of PSII was maintained better in
of GSII. The high levels of GSII in the stay- the stay-green plants under water-limited
green sorghums might have enhanced the re- conditions compared to R16 plants. Since
assimilation of N from photorespiration, even chlorophyll is required for the integration of
though photorespiration is supposed to be low newly synthesised D1 protein (Mullet et al.,
in C4 plants (Makino et al., 2003). 1990), the loss of chlorophyll in the WL R16
plants might have prevented the integration of
Chloroplast proteins. Abundance of LHCPII newly synthesised D1 protein (if any was
differed very little between the WW and WL synthesised).
plants of the stay-green genotypes (B35 and Cyt f is involved in transferring electrons
KS19) (Table 2); but was reduced by water- from plastoquinone (PQ) to Plastocyanin (PC);
limitation in E36-1 and R16 plants. The while Fd transfers electrons from PSI to
reduction in LHCPII between anthesis and NADP. Cyt f and Fd (results not shown) were
maturity was also higher in E36-1 (56%) and also retained throughout in the stay-green
R16 (53%) compared with B35 (10.3%) and genotypes (B35 and KS19) under water
KS19 (5.1%). LHCPII is a major contributor limitation, unlike in the E36-1 and R16
to the overall loss of protein during leaf genotypes. Davies et al. (1990) have reported
senescence (Matile, 1992). In stay-green that Cyt f is more stable in stay-greens than in
genotypes, LHCPII remains stabilised and senescent genotypes. The reductions of Cyt f
proteolytic cleavage is restricted due to a small and Fd proteins in the WL E36-1 and R16
N-terminal that protrudes into the stoma genotypes could also result in reduced electron
(Thomas and Donnison, 2000). transport and overall photosynthesis rates.
The 33 kDa oxygen-evolving complex Indeed, in antisense lines with reduced Fd,
protein (OEC33) is known to stabilise the Holtgrefe et al. (2003) found that cyclic
catalytic Mn cluster, which is essential for electron transport, determined by quantum
water oxidation (Zhang et al., 1998). A yields of PSI and PSII, was enhanced,
reduction in or degradation of OEC33, as a
672 I.K.A. GALYUON et al.
whereas CO2 assimilation rate in some lines The reduction in the abundance of PEPC
showed photoinhibition. and PEPCK in the WL R16 plants could have
impaired the production of malate needed for
Photosynthetic enzymes. Phosphoenol CO2 production in the bundle sheath cells and,
pyruvate carboxylase (PEPC) was also consequently, CO2 assimilation rate in these
maintained at higher levels in the stay-green plants.
plants (B35 and KS19) compared to E36-1 and The abundance of rubisco in leaves is
R16 under water limitation (Table 2). The controlled by the rate of its synthesis and
availability of functional PEPCK in the WW degradation (Parry et al., 2002). The
and WL stay-green plants could have resulted abundance of the large subunit (LSU) of
in better maintenance of photosynthesis than rubisco in all the genotypes was not affected
in E36-1 and R16 under water-limiting by water limitation throughout the sampling
conditions. Photosynthesis in C 4 plants, period. Observations on the effect of drought
including sorghum, involves the enzymes on rubisco (LSU) are varied (Zasheva et al.,
PEPCK, PEPC and Rubisco, among others. 2009). In some studies, it has been found that
PEPC occupies a key position as the initial CO2- drought has no effect on rubisco (Pellox et
fixing enzyme of the C4 pathway, and is a major al., 2001) whereas in other studies drought
control point in this pathway (Liu et al., 2017). resulted in reduction of the large subunit of
The activity of PEPC is regulated at many the enzyme (Inmaculada et al., 2006; Zasheva
levels, including the phosphorylation catalysed et al., 2009). Therefore, our findings are
by phosphoenolpyruvate carboxylase kinase similar to those reported Pelloux et al. (2001).
(PEPCK) (Monreal et al., 2013). The
phosphorylation of PEPC catalysed by PEPCK Grain yield. The present study has shown
enables PEPC to perform its catalytic that maintenance of GLA does not result in
activities. Therefore, under WL conditions, the lower grain yields due to poor sink strength.
42% reduction in PEPCK (Jeanneau et al., This is in agreement with findings obtained
2002) activity could have resulted in the from field studies (Borrell et al., 2000b; Borrell
deactivation of PEPC in R16 (60% reduction). et al., 2014ab; Jordan et al., 2012). Panicle
Blocking of PEPCK synthesis in the leaf of harvest index (PHI) was maintained in B35,
maize and sorghum leads to marked inhibition E36-1 and KS19 under water limitation;
of CO 2 assimilation, not due to stomatal whereas in R16 it was significantly (P<0.05)
closure, but as a consequence of a decrease reduced,. These differences in PHI are due,
in other photo-activated C4 cycle enzymes and in part, to the lower seed set in WL R16
perturbation of the Benson-Calvin cycle compared with the other genotypes. Similarly,
(Jeanneau et al., 2002). Therefore, current seed size, measured as mean 100 seed weight,
findings support the reports that was maintained in B35 and KS19, but reduced
phosphorylation of PEPC by PEPCK is in WL E36-1 and R16 plants. Even in E36-1, it
fundamental for CO2 assimilation in the C4 was apparent that seed size was less reduced
photosynthetic pathway (Jeanneau et al., compared to R16 by water limitation.
2002). The reduction in the amounts of both The stay-green sorghum genotypes,
PEPC and PEPCK, could partially cause particularly B35 and KS19, exhibit delayed leaf
reduced CO2 assimilation rate in the WL R16 senescence and maintain photosynthesis for
plants. In the same vein, the high CO 2 longer periods as a result of maintenance of
assimilation rates observed in the stay-green the integrity of the photosynthetic apparatus,
plants, particularly B35, were likely associated chlorophyll biosynthesis and turn over, as well
with the retention of high amounts and activities as photosynthetic enzymes under drought
of PEPC and PEPCK under water limitation. stress. Furthermore, the onset of senescence
 Physiological and biochemical basis for stay-green trait in sorghum 673
is delayed and/or the rate of senescence is Borrell, A.K., Hammer, G.L. and Douglas,
reduced in stay-green (B35 and KS19) A.C.L. 2000b. Does maintaining green leaf
compared with senescent genotype. E36-1 does area in sorghum improve yield under
not behave as B35 or KS19, confirming drought? II. Dry matter production and
sorghum plants may stay green differently. yield. Crop Science 40:1037-1048.
Borrell, A.K. and Hammer, G. L. 2000. Nitrogen
ACKNOWLEDGEMENT dynamics and the physiological basis of
stay-green in sorghum. Crop Science
Funding for this study was provided by the 40:1295-1307.
Association of Commonwealth Universities, Borrell, A.K., Bidinger, F.R., Tao, Y.Z.,
arranged through University of Cape Coast, McIntyre, C.L. and Douglas A.C.L. 2001.
Ghana, and Aberystwyth University, Wales, Physiological and molecular aspects of
United Kingdom. stay-green in sorghum. In: Borrell, A.K. and
Henzell, R.G. (Eds.). Proceedings from the
REFERENCES Fourth Australian Sorghum Conference,
Kooralbyn, 5-8 February 2001. CD-rom
Anjum, S. A., Xie, X. Y., Wang, L. C., Saleem, format. Range Media Pty Ltd. ISBN:0-
M. F., Man, C. and Lei, W. 2011. 7242-2163-8.
Morphological, physiological and Borrell, A.K., Mullet, J.E., George-Jaeggli, B.,
biochemical responses of plants to drought van Oosterom, E.J., Hammer, G.L., Klein,
stress. African Journal Agricultural P.E. and Jordan, D.R. 2014a. Drought
Research 6:2026–2032. adaptation of stay-green sorghum is
Badigannavar, A., Teme, N., Costa de Oliveira associated with canopy development, leaf
A., Li, G., Vaksmann, M., Vianna, V. E. and anatomy, root growth, and water uptake.
Ganapathi, T. R. 2018. Physiological, Journal of Experimental Botany
genetic and molecular basis of drought 65(21):6251-6263. doi:10.1093/jxb/
resilience in sorghum [Sorghum bicolor (L.) eru232.
Moench]. Indian Journal of Plant Borrell, A.K., van Oosterom, E.J., Mullet, J.E.,
Physiology 23(4):670-688. George-Jaeggli, B., Jordan, D.R., Klein,
Bidinger, F.R., Mahalakshmi. V. and Rao, P.E. and Hammer, G.L. 2014b. Stay-green
G.D.P. 1987. Assessment of drought alleles individually enhance grain yield in
resistance in pearl millet (Pennisetum sorghum under drought by modifying
americanum (L.) Leeke). 1. Factors canopy development and water uptake
affecting yield under stress. Australian patterns. New Phytologist 203:817–830
Journal of Agricultural Research 38:37-48. (doi: 10.1111/nph.12869).
Borrell, A.K., Bidinger, F.R. and Sunitha, K. Boyles, R.E., Breton, Z.W. and Kresovich, S.
1999. Stay-green trait associated with yield 2019. Genetic and genomic resources of
in recombinant inbred sorghum lines sorghum to connect genotype with
varying in rate of leaf senescence. phenotype in contrasting environments. The
International Sorghum and Millets Plant Journal 97:19-39.
Newsletter 40:31-34. Burgess, M.G., Rush, C.M., Piccinni, G. and
Borrell, A.K., Hammer, G.L. and Douglas, Schuster, G. 2002. Relationship between
A.C.L. 2000a. Does maintaining green leaf charcoal rot, the stay-green trait, and
area in sorghum improve yield under irrigation in grain sorghum. Phytopathology
drought? I. Leaf growth and senescence. 92, S10.
Crop Science 40:1026-1037.
674 I.K.A. GALYUON et al.
Crasta, O.R., Xu, W.W., Rosenow, D.T., at the luminal side of photosystem Ii in
Mullet, J.E. and Nguyen, H.T. 1999. spinach chloroplasts: Evidence from light-
Mapping of post-flowering drought induced cross-linking of proteins in the
resistance traits in sorghum: association donor-side photoinhibition. Plant Cell
between QTLs influencing premature Physiology 44:451-456.
senescence and maturity. Molecular and Hilditch, P. I., Thomas, H., Thomas, B. J. and
General Genetics 262:579-588. Rogers, L. J. 1989. Leaf senescence in non-
Czarnecki, O. and Grimm, B. 2012. Post- yellowing mutant of Festuca pratensis:
translational control of tetrapyrrole proteins of photosystem II. Planta
biosynthesis in plants, algae and 177:265-272.
cyanobacteria. Journal of Experimental Holtgrefe, S., Bader, K. P., Horton, P., Scheibe,
Botany 63(4):1675-1687. R., von Schaewen, A. and Backhausen, J.
Davies, T.G.E., Thomas, H., Thomas, B.J. and E. 2003. Decreased content of leaf
Rogers, L.J. 1990. Leaf senescence in non- ferredoxin changes electron distribution and
yellowing mutant of Festuca pratensis: limits photosynthesis in transgenic potato
metabolism of cytochrome f. Plant plants. Plant Physiology 133:1768-1778.
Physiology 93:588-595. Igamberdiev, A.U., Eprintsev, A.T., Ferdorin,
de Villers, L., Turk, K., Thomas, H. and and Popov, V.N. 2014. Phytochrome-
Howarth, C. 1993. Analysis and exploitation mediated regulation of plant respirationand
of the stay-green character in sorghum. photorespiration. Plant Cell and
Overseas Development Administration Environment 37:290-299.
Project R4885 Annual Report. Inmaculada, J., Navarro, R.M., Lenz, C.,
Demirevska, K., Zasheva, D., Dimitrov, R., Ariza, D. and Jorrin, J. 2006. Variation in
Simova-Stoilova, L., Stamenova, M. and the holm oak leaf proteome at plant
Feller, U. 2009. Drought effects on developmental stages, between
Rubisco in wheat: Changes in Rubisco large provenances and in response to drought
subunit. Acta Physiologiae Plantarum stress. Proteomics 6S207-S214.
31:1129-1138. doi:10.1007/s11738-009- doi:10.1002/pmic.
0331-2. Jeanneau, M., Vidal, J., Gousset-Dupont, A.,
Escalada, R.G. and Plucknett, D.L. 1975. Lebouteiller, B., Hodges, M., Gerentes, D.
Ratoon cropping of sorghum: II. Effect of and Perez, P. 2002. Manipulating PEPC
daylength and temperature on tillering and levels in plants. Journal of Experimental
plant development. Agronomy Journal Botany 53:1837-1845.
67:479-484. Jordan, D.R., Hunt, C.H., Cruickshank, A.W.,
FAO. 2014. FAOSTAT http://faostat.fao.org. Borrell, A. K. and Henzell, R. G. 2012. The
(Accessed 26th April 2017). relationship between the stay-green trait
Harris, K., Subudhi, P. K., Borrell, A., Jordan, and grain yield in elite sorghum hybrids
D., Rosenow, D., Nguyen, H., Klein, P., grown in a range of environments. Crop
Klein, R. and Mullet, J. 2007. Sorghum Science 52:1153–1161.
stay-green QTL individually reduce post- Laemmli, U.K. 1970. Cleavage of structural
flowering drought-induced leaf proteins during the assembly of the head
senescence. Journal of Experimental of the bacteriophage T4. Nature 227:680-
Botany 58:327-338. 685.
Henmi, T. Yamasaki, H., Sakuma, S., Liu, X., Li, X., Zhang, C., Dai, C., Zhou, J.,
Tomokawa, Y., Tamura, N., Shen, J-R. and Ren. C. 2017. Phosphoenolpyruvate
Yamamoto, Y. 2003. Dynamic interaction carboxylase regulation in C4-PEPC-
between the D1 protein, CP43 and OEC33 expressing transgenic rice during early
 Physiological and biochemical basis for stay-green trait in sorghum 675
responses to drought stress. Physiologia Oliveira, I.C., Brears, T., Knight, T.J., Clark,
Plantarum 159:178-200. A. and Coruzzi, G.M. 2002.
Mahalakshmi, V. and Bidinger, F.R. 2002. Overexpression of cytosolic glutamine
Evaluation of the stay-green germplasm synthetase. Relation to nitrogen, light, and
lines at ICRISAT. Crop Science 42:965-974. photorespiration. Plant Physiology
Makino, A., Sakuma, H., Sudo, E. and Mae, 129:1170-1180.
T. 2003. Differences between maize and Parry, M.A.J., Andralojc, P.J., Khan, S., Lea,
rice in N-use efficiency for photosynthesis P.J. and Keys, A.J. 2002. Rubisco activity:
and protein allocation. Plant Cell Effects of drought stress. Annals of
Physiology 44:952-956. Botany 89:833-839.
Matile, P. 1992. Chloroplast senescence. In: Paterson, A.H., Bowers, J.E., Feltus, F.A.,
Baker, N. R. and Thomas, H (eds) Crop Tang, H.B., Lin, L.F. and Wang, X.Y. 2009.
Photosynthesis: spatial and temporal Comparative genomics of grasses promises
determinants. Elsevier, Amsterdam. pp. a bountiful harvest. Plant Physiology
413-440. 149:125-131.
Maxwell, K. and Johnson, G.N. 2000. Pelloux, J., Jolivet, Y., Fontaine, V., Banvoy,
Chlorophyll fluorescence - A practical guide. J. and Dizengremel, P. 2001. Changes in
Journal of Experimental Botany 51:659- Rubisco and Rubisco activase gene
668. expression and polypeptide content in Pinus
Miyake, C., Miyata, M., Shinzaki, Y. and halepensis M. subjected to ozone and
Tomizawa, K. 2005. CO 2 response of drought. Plant Cell and Environment
cyclic electron flow around PSI (CEF-PSI) 24:671-684. doi:10.1046/j.1365-3040.
in tobacco leaves – Relative electron fluxes 2001.00665.x.
through PSI and PSII determine the Peng, Y., Li, C. and Fritschi, F.B. 2013.
magnitude of non-photochemical Apoplastic infusion of sucrose into stem
quenching (NPQ) of Chl fluorescence. internodes during female flowering does
Plant Cell Physiology 46:629-637. not increase grain yield in maize plants
Monreal, J.A., Arias-Baldrich, C., Pérez- grown under nitrogen-limiting conditions.
Montaño, F., Grandullo, J., Echevarría, C., Physiologia Plantarum 148:70-80.
Garcia-Mauriño. S. 2013. Factors involved Ronchi, C.P., DaMatta, F. M., Batista, K.D.,
in the rise of phosphoenolpyruvate Moraes, G.A.B.K., Loureiro, M.E. and
carboxylase-kinase activity caused by Ducatti, C. 2006. Growth and
salinity in sorghum leaves. Planta photosynthetic down-regulation in Coffea
237:1401-1413. arabica in response to restricted root
Mullet, J.E., Gamble-Klein, P. and Klein, R.R. volume. Functional Plant Biology 33,
1990. Chlorophyll regulates accumulation 1013–1023. (doi:10.1071/FP06147).
of plastid-encoded chlorophyll apoproteins Rosenow, D.T., Clark, L.E., Dahlberg, J.A.,
CP43 and D1 by increasing apoprotein Frederiksen, R.A., Odvody, G.N.,
stability. Proceedings of the National Peterson, G.C., Schaefer, K., Collins, S.D.,
Academy of Sciences, USA 87:4038-4042. Jones, J.W. and Hamburger, A.J. 2002.
Németh, E., Nagy, Z. and Pécsváradi, A. 2018. Release of four A/B sorghum parental lines
Chloroplast glutamine synthetase, the key ATx642 through ATx645. International
regulator of nitrogen metabolism in wheat, Sorghum and Millets Newsletter 43:24–30.
performs its role by fine regulation of Rosenow, D.T., Quisenberry, J.E., Wendt,
enzyme activity via negative cooperativity C.W. and Clark, L.E. 1983. Drought
of its subunits. Frontiers in Plant Science tolerant sorghum and cotton germplasm.
9:1-10. Doi:10.3389/fpls.2018.00191.
676 I.K.A. GALYUON et al.
Agricultural Water Management 7:207- Thomas, H., Thomas, H. M. and Ougham, H.
222. 2000. Annuality, perenniality and cell death.
Smart, C.M., Hosken, S.E., Thomas, H., Journal of Experimental Botany
Greaves, J.A., Blair, B.G., Schuch, W. 51(352):1781-1788.
1995. The timing of maize leaf senescence Tsuji, W., Ali, M.E.K., Inanaga, S. and
and characterisation of senescence-related Sugimoto, Y. 2003. Growth and gas
cDNAs. Physiologia Plantarum 93:673– exchange of three sorghum cultivars under
682. drought stress. Biologia Plantarum 46:583-
Srinivas, G., Satish, K., Murali Mohan, S., 587.
Nagaraja Reddy, R., Madhusudhana, R., Vadez, V., Deshpande, S.P., Kholova, J.,
Balakrishna, D., Venkatesh Bhat, B., Hammer, G.L., Borrell, A. K., Talwar, H.S.
Howarth, C.J. and Seetharama, N. 2008. and Hash, C.T. 2011. Stay-green
Development of genic-microsatellite quantitative trait loci’s effects on water
markers for sorghum staygreen QTL using extraction, transpiration efficiency and seed
a comparative genomic approach with rice. yield depend on recipient parent
Theoretical and Applied Genetics 117:283– background. Functional Plant Biology
296. 38:553–566. (doi:10.1071/ FP11073).
Tao, Y. Z., Henzell, R.G., Jordan, D.R., Butler, Van Oosterom, E.J., Jayachandran, R. and
D.G., Kelly, A.M. and McIntyre, C.L. Bidinger, F. 1996. Diallel analysis of
2000. Identification of genomic regions staygreen trait and its components in
associated with stay-green in sorghum by sorghum. Crop Science 36:540-555.
testing RILs in multiple environments. Vavillin, D.V. and Vermaas, F.J. 2002.
Theoretical and Applied Genetics Regulation of the tetrapyrrole biosynthetic
100:1225-1232. pathway leading to heme and chlorophyll
Thomas, H. and Donnison, I. 2000. Back from in plants and cyanobacteria. Plant
the brink: plant senescence and its Physiology 115:9-24.
reversibility. In: Bryant, J.A., Hughes, S.G. Walulu, R.S., Rosenow, D.T., Wester, D.B.
and Garland, J.M. (Eds.). Programme cell and Nguyen, H. T. 1994. Inheritance of
death in animals and plants. Oxford: Bios. stay-green trait in sorghum. Crop Science
pp. 149-162. 34:970-972.
Thomas, H. and Howarth, C J. 2000. Five Wolfe, D.W., Henderson, D.W., Hsiao, T.C.
ways to stay green. Journal of and Alvino, A. 1988. Interactive water and
Experimental Botany 51: 329-337. nitrogen effects on senescence of maize.
Thomas, H., Morgan, W.G., Thomas, A.M. I. Leaf area duration, nitrogen distribution
and Ougham, H.J. 1999. Expression of the and yield. Agronomy Journal 80:859-864.
stay-green character introgressed into Xu, W., Rosenow, D.T. and Nguyen, H.T.
Lolium temulentum Ceres from a 2000. Stay-green trait in grain sorghum:
senescence mutant of Festuca pratensis. relationship between visual rating and leaf
Theoretical and Applied Genetics 99:92– chlorophyll concentration. Plant Breeding
99. 119:365-367.
Thomas, H. and Ougham, H. 2014. The stay- Yang, T.Y., Zhu, L.N., Wang, S.P., Gu, W.J.,
green trait. Journal of Experimental Botany Huang, D.F., Xu, W.P., Jiang, A.L. and Li,
65(14):3889-3900. (doi:10.1093/jxb/ S.C. 2007. Nitrate uptake kinetics of
eru037). grapevine under root restriction. Scientia
Thomas, H. and Rogers, L.J. 1990. Turning Horticulturae 111: 358-364.
over an old leaf. University of Wales Yang, Z., Hammer, G., van Oosterom, E.,
Reviews of Science and Technology 6:29- Rochas, D., and Deifel, K. 2010. Effects
38.
 Physiological and biochemical basis for stay-green trait in sorghum 677
of pot size on growth of maize and protein of photosystem II studied by
sorghum plants. In: George-Jaeggli, B., Fourier transform infrared spectroscopy.
Jordan, D.J. (Eds.). 1st Australian Summer FEBS Letters 426:347-351.
Grains Conference. Gold Coast, Australia: Zhu, L.N., Wang, S.P., Yang, T.Y., Zhang,
Grains Research and Development C.X. and Xu, W.P. 2006. Vine growth and
Corporation. nitrogen metabolism of ‘Fujiminori’
Zhang, H., Ishikawa, Y., Yamamoto, Y. and grapevines in response to root restriction.
Carpentier, R. 1998. Secondary structure Scientia Horticulturae 107:143-149.
and thermal stability of the extrinsic 23 kDa