Step-by-Step Instructions for Reading a Primary Research Article

1. Begin by reading the introduction, not the abstract.

The abstract is that dense first paragraph at the very beginning of a paper. In fact, that’s often the only part of a paper that many non-scientists read when they’re trying to build a scientific argument. (This is a terrible practice. Don’t do it.) I always read the abstract last, because it contains a succinct summary of the entire paper, and I’m concerned about inadvertently becoming biased by the authors’ interpretation of the results. 

2. Identify the big question.

Not “What is this paper about?” but “What problem is this entire field trying to solve?” This helps you focus on why this research is being done.  Look closely for evidence of agenda-motivated research.

3. Summarize the background in five sentences or less.

What work has been done before in this field to answer the big question? What are the limitations of that work? What, according to the authors, needs to be done next? You need to be able to succinctly explain why this research has been done in order to understand it.

4. Identify the specific question(s).

What exactly are the authors trying to answer with their research? There may be multiple questions, or just one. Write them down.  If it’s the kind of research that tests one or more

null hypotheses

, identify it/them.

5. Identify the approach.

What are the authors going to do to answer the specific question(s)?

6. Read the methods section.

Draw a diagram for each experiment, showing exactly what the authors did. Include as much detail as you need to fully understand the work. 

7. Read the results section.

Write one or more paragraphs to summarize the results for each experiment, each figure, and each table. Don’t yet try to decide what the results mean; just write down what they are. You’ll often find that results are summarized in the figures and tables. Pay careful attention to them!  You may also need to go to supplementary online information files to find some of the results. Also pay attention to:

• The words “significant” and “non-significant.” These have precise statistical meanings. Read more about this here.
• Graphs. Do they have error bars on them? For certain types of studies, a lack of confidence intervals is a major red flag.
• The sample size. Has the study been conducted on 10 people, or 10,000 people? For some research purposes a sample size of 10 is sufficient, but for most studies larger is better.

8. Determine whether the results answer the specific question(s).

What do you think they mean? Don’t move on until you have thought about this. It’s OK to change your mind in light of the authors’ interpretation — in fact, you probably will if you’re still a beginner at this kind of analysis — but it’s a really good habit to start forming your own interpretations before you read those of others.

9. Read the conclusion/discussion/interpretation section.

What do the authors think the results mean? Do you agree with them? Can you come up with any alternative way of interpreting them? Do the authors identify any weaknesses in their own study? Do you see any that the authors missed? (Don’t assume they’re infallible!) What do they propose to do as a next step? Do you agree with that?

10. Go back to the beginning and read the abstract.

Does it match what the authors said in the paper? Does it fit with your interpretation of the paper?

11. Find out what other researchers say about the paper.

Who are the (acknowledged or self-proclaimed) experts in this particular field? Do they have criticisms of the study that you haven’t thought of, or do they generally support it? Don’t neglect to do this! Here’s a place where I do recommend you use Google! But do it last, so you are better prepared to think critically about what other people say.

Soil Science and Plant Nutrition (2008) 54, 253–258 doi: 10.1111/j.1747-0765.2007.00234.x

© 2008 Japanese Society of Soil Science and Plant Nutrition

Blackwell Publishing Ltd

ORIGINAL ARTICLE
Temperature and photosynthesis in rose shootsORIGINAL ARTICLE

Effects of temperature on photosynthesis and plant growth in
the assimilation shoots of a rose

Ayuko USHIO1,2, Tadahiko MAE2 and Amane MAKINO2
1National Institute of Floricultural Science, National Agriculture and Food Research Organization, Ibaraki 305-8519 and
2Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

Abstract

The effects of temperature on photosynthesis, ribulose-bisphosphate carboxylase (Rubisco) content and
whole plant growth were investigated in the assimilation shoots of a rose (Rosa hybrida L.). Assimilation
shoots were grown at two different day/night temperature regimes of 20/15°C (LT) and 30/25°C (HT) for
42 days after 1-month growth. Although LT initially suppressed the photosynthetic rate during the first
7 days, prolonged growth at LT enhanced potential photosynthesis. This was associated with increases in
Rubisco and N contents at the level of a single leaf. Rubisco content and the photosynthetic rate at 25°C
were 2.8-fold and 1.6-fold higher in the LT plants than in the HT plants at day 42, respectively. The relative
growth rate at the level of the whole plant was lower in the LT plants during the first 28 days and the leaf
area ratio was smaller in the LT plants throughout the experiment. However, enhanced photosynthesis
during growth at LT led to increases in the net assimilation rate at the level of the whole plant, and final
biomass at day 42 did not differ between the two temperature treatments. To enhance the photosynthetic
capacity in assimilation shoots of a rose, cultivation at 20/15°C is better than cultivation at 30/25°C.

Key words: biomass, growth rate, photosynthesis, Rosa hybrida L., Rubisco, temperature.

INTRODUCTION

Roses are one of the most popular plants in greenhouse
cultivation. In rose cultivation, the basal shoots emerging
in the early stage of growth are artificially bent down as
assimilation shoots to effectively catch sunlight, and
then new shoots emerging successively are harvested
as cut flowers for rose products (Okawa and Suematsu
1999). This “shoot-bending” (or “arching”) cultivation
technique leads to a higher yield and high quality of
the flowering shoot (Okawa and Suematsu 1999). This
technique makes it possible that higher amounts of
carbohydrate are transported to flowering shoots from
the leaves of bent shoots.

To attain high yields of cut flowers using the shoot-
bending technique, a few important approaches are
proposed. One is to optimize the leaf area index (LAI) of
the bent-shoot canopies, and the others are to enhance

the photosynthetic capacity of the assimilation shoots
before shoot bending and to maintain a high photosyn-
thetic capacity in the shoots after bending. For example,
it is proposed that an LAI of 3.0 should be optimal for the
production of the shoot-bending technique (Shimomura
et al. 2003). However, it is not known how photosynthesis
can be enhanced in the assimilation shoots before bending
and how it can be maintained at a high capacity after
bending. Many cultural environments, such as nutrition,
temperature and sunlight, may affect the photosynthetic
capacity of rose leaves. As roses are generally grown in
soil-less culture under sufficient nutritional conditions,
nutrition does not limit plant growth and the photosyn-
thetic capacity of the leaves. In conventional greenhouse
cultivation, temperature largely changes throughout
the year, and the difference in average temperature in a
greenhouse between summer and winter is approximately
10°C. However, it is unknown how temperature affects
photosynthesis and rose growth.

In the present study, we examined the effects of growth
temperature on the photosynthetic capacity at a single
leaf level in the assimilation shoots of rose before shoot
bending. The uppermost, young leaf was used throughout
the experimental period. As roses are generally cultivated
over a range of 20 –30°C for day temperature and 18 –20°C

Correspondence: A. MAKINO, Graduate School of
Agricultural Science, Tohoku University, Tsutsumidori-
Amamiyamachi, Sendai 981-8555, Japan. Email: makino@
biochem.tohoku.ac.jp
Received 6 July 2007.
Accepted for publication 4 November 2007.

254 A. Ushio et al.

© 2008 Japanese Society of Soil Science and Plant Nutrition

for night temperature (Beeson 1990; Bredmose 1998;
Gonzalez-Real and Baille 2000; Jiao and Grodzinski
1998; Kim and Lieth 2003; Kool et al. 1996), we grew
the assimilation shoots at two different day/night
temperature regimes of 20/15°C and 30/25°C. We analyzed
the difference in the growth rate of the assimilation shoots
under these different temperatures.

MATERIALS AND METHODS

Plant culture
Rosa hybrida L. cv. Asami Red (Roterose) plants were
used. Three rooted single-node cuttings with a five-leaflet
leaf grown on rockwool blocks (5 cm × 5 cm × 5 cm)
were planted on a rockwool plate (30 cm length × 20 cm
width × 7.5 cm height). The rooted cutting plants had a
5– 6 cm new shoot. Two plates were placed in a plastic
container. The plants were cultivated in a greenhouse
at Tsukuba, Japan. The plants were fertilized with a
nutrient solution containing 0.3 mmol L–1 KH2PO4,
0.5 mmol L–1 MgSO4, 1 mmol L

–1 CaCl2, 1 mmol L
–1 KCl,

0.5 mmol L–1 NH4NO3, 1 mmol L
–1 KNO3, 50 μmol L

–1

Fe-ethylenediaminetetraacetic acid, 50 μmol L–1 H3BO3,
9 μmol L–1 MnSO4, 0.3 μmol L

–1 CuSO4, 0.8 μmol L
–1

ZnSO4 and 0.1 μmol L
–1 Na2MoO4. The nutrient solution

(2 L per plastic container) was renewed once (0 –2 weeks
after plantation) or twice (2– 4 weeks after plantation)
per week. When the solution was renewed the pH was
adjusted to 5.3 with HCl.

After 1 month, the plants (approximately 25 cm shoot
length and 5–8 five-leaflet leaves) were transferred to
temperature-controlled growth chambers operating at
two different day/night temperature regimes of 20/15°C
(LT) and 30/25°C (HT) under natural sunlight condi-
tions. The plants were fertilized with the same nutrient
solution. The nutrient solution (5 L per plastic container)
was renewed twice per week, and the pH was adjusted
to 5.3 with HCl. The plants were grown for 42 days and
all rose buds were removed throughout the experimental
period.

Growth analysis
Plants were harvested every 2 weeks between day 0 and
day 42 after transfer to different growth temperatures.
The harvested plants were divided into leaves and stems.
The leaf area was measured. The leaves and stems were
oven-dried at 80°C for more than 3 days. The rockwool
plates were oven-dried at 80°C for more than 7 days,
and then the roots were carefully picked out with
tweezers. The leaves, stems and roots were weighed and
milled. Relative growth rate (RGR), net assimilation
rate (NAR), leaf area ratio (LAR), leaf weight ratio
(LWR) and specific leaf area (SLA) were calculated
from total dry weight and leaf area.

Determination of chlorophyll, Rubisco and
total leaf N in a leaf
The uppermost, young leaf was cut at day 0, 14 and 42
after transfer to the different growth temperatures and
stored at −80°C. The chlorophyll (Chl) and total leaf
N contents were determined according to the method
of Makino and Osmond (1991), except that a buffer
of 100 mmol L–1 Na-phosphate, pH 7.5, 0.8% (v/v) 2-
mercaptoethanol, 4 mmol L–1 iodoacetic acid and 20%
(v/v) glycerol was used.

Rubisco contents were also determined according to
the method of Makino and Osmond (1991) with some
modifications. One leaf was powdered in liquid N2 in a
mortar with a pestle and sea sand, and then homogenized
in 100 mmol L–1 Na-phosphate buffer, pH 7.5, containing
0.8% (v/v) 2-mercaptoethanol, 4 mmol L–1 iodoacetic acid,
20% (v/v) glycerol and 2% (w/v) polyvinylpyroridone.
The homogenate was treated with a lithium dodecyl-
sulfate solution (4% [w/v] final concentration) and 2-
mercaptoethanol (2% [v/v] final concentration) at 100°C
for 90 s. After centrifugation at 10,000 g for 8 min the
supernatant fluid was stored at −30°C until analysis by
sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).

Gas exchange measurements
Gas exchange rates were measured at the level of a single
leaf with a LI-6400 portable photosynthesis system
(Li-Cor, Lincoln, NE, USA). All measurements were
made at a photosynthetical photon flux density (PPFD)
of 1,000 μmol quanta m−2 s−1 (10% blue light-emitting
diodes [LEDs] in red LEDs), a leaf-to-air vapor pressure
difference of 1.0–1.2 kPa and a partial pressure of CO2
of 37 Pa between 09:00 and 12:00 hours. To measure
the rates at growth temperature, the rates were measured
at a leaf temperature of 20°C for the LT plants and at
30°C for the HT plants, respectively. A young, expanding
leaf was used for the measurements between day 0 and
day 10 after transfer to the different growth temperatures,
and then a young, fully expanded leaf was used between
day 14 and day 42. In addition, the rates at a leaf
temperature of 25°C were also measured for both
treatments.

RESULTS

Photosynthesis, total N and Rubisco contents
in a leaf

Changes in the rates of photosynthesis measured at
growth temperature are shown in Fig. 1. The photosyn-
thetic rate was higher in the HT plants than in the LT
plants during the first 7 days, and then the rates were

Temperature and photosynthesis in rose shoots 255

© 2008 Japanese Society of Soil Science and Plant Nutrition

not significantly different between day 10 and day 18
after transfer to the different growth temperatures.
However, the photosynthetic rate at day 42 was sig-
nificantly higher in the LT plants than in the HT plants
(P < 0.01). Table 1 shows the rates of CO2 assimilation measured at the same leaf temperature of 25°C. The rates at 25°C were slightly higher at day 14 in the LT plants, and the difference became greater at day 42. Stomatal conductance tended to show a similar response to that of photosynthesis. No difference in the intercellular

CO2 partial pressure was found at day 14, but it was
higher in the HT plants at day 42.

Table 2 shows the changes in total N, Chl and Rubisco
contents. Total leaf N and Rubisco contents were slightly
greater in LT plants at day 14. At day 42, all parameters
were markedly higher in the LT plants than in the HT
plants. Among them, the Rubisco content was 2.8-fold
greater in the LT plants. Thus, all parameters increased
in the LT plants over the experimental period, and
decreased in the HT plants after day 14.

Plant growth
Table 3 shows the changes in plant mass at the whole
plant level, the dry weight of each organ, and the total
leaf area. The total plant mass was greater in the HT
plants than in the LT plants between day 0 and day 28,
but the final biomass at day 42 was not significantly
different. The total leaf area per plant was greater in the
HT plants than in the LT plants throughout the experi-
mental period. The root biomass was not different
between the LT and HT plants.

The growth rate was analyzed at the level of the
whole plant (Fig. 2). The RGR, which is defined as the

Figure 1 Changes in the photosynthetic rate measured at
growth temperature after transfer to two different day/night
temperatures of 20/15°C (�) and 30/25°C (�). Measurements
were made at a photosynthetical photon flux density of
1,000 μmol quanta m−2 s−1 (10% blue light-emitting diodes
[LEDs] in red LEDs), a leaf-to-air vapor pressure difference of
1.0–1.2 kPa and a partial pressure of CO2 of 37 Pa. Each point
represents the mean ± standard deviation of the measurements
on the different leaves (n = 6, 8, 6, 11, 5, 3 and 17 at day 5, 7,
10, 14, 16, 18 and 42 for 20/15°C [LT] plants, respectively;
n = 5, 7, 7, 7, 5, 4 and 20 at day 5, 7, 10, 14, 16, 18 and 42
for 30/25°C [HT] plants, respectively). The data were fitted
with a second-order polynomial between day 5 and day 18.
*P < 0.05; **P < 0.01.

Table 1 Photosynthetic rate, stomatal conductance and intercellular CO2 partial pressure measured at 25°C in a young rose leaf
after transfer to two different temperatures of 20/15°C and 30/25°C (day/night)

Day
Photosynthesis

(CO2 μmol m
−2 s−1)

Stomatal conductance
(mol m−2 s−1)

Intercellular
CO2 (Pa)

14 30/25°C 18.3 ± 1.4
*

0.35 ± 0.06 27.3 ± 1.4
20/15°C 20.3 ± 2.1 0.37 ± 0.05 26.7 ± 1.4

42 30/25°C 15.5 ± 1.7
**

0.24 ± 0.03
**

24.6 ± 1.5
**20/15°C 24.8 ± 1.3 0.30 ± 0.03 22.1 ± 1.3

Data are mean ± standard deviation (n = 5–14). *P < 0.05; **P < 0.01.

Table 2 Total leaf N, chlorophyll and Rubisco contents in a
young rose leaf after transfer to two different temperatures of
20/15°C and 30/25°C (day/night)

Day
Total N

(mmol m−2)
Chl

(mmol m−2)
Rubisco
(g m−2)

0 112 ± 20
*

0.36 ± 0.04 1.8 ± 0.1
14 30/25°C 139 ± 7 0.65 ± 0.05 3.7 ± 0.1

20/15°C 171 ± 10 0.68 ± 0.03 4.5 ± 0.6
42 30/25°C 104 ± 9

**
0.45 ± 0.01

**
2.0 ± 0.1

**20/15°C 187 ± 31 0.75 ± 0.11 5.5 ± 0.3

Data are mean ± standard deviation (n = 3 – 4). *P < 0.05; **P < 0.01. Chl, chlorophyll.

256 A. Ushio et al.

© 2008 Japanese Society of Soil Science and Plant Nutrition

Table 3 Total plant mass, dry weight of each organ and total leaf area of rose after transfer to two different temperatures of 20/
15°C and 30/25°C (day/night)

Day
Total dry
weight (g)

Leaf dry
weight (g)

Stem dry
weight (g)

Root dry
weight (g)

Leaf area
(m2 plant−1)

0 1.41 ± 0.16 0.53 ± 0.11 0.40 ± 0.08 0.48 ± 0.04 0.013 ± 0.003
14 30/25°C 3.45 ± 0.24

**
2.16 ± 0.18

**
0.80 ± 0.12

**
0.50 ± 0.10 0.049 ± 0.009

**20/15°C 2.68 ± 0.46 1.42 ± 0.40 0.61 ± 0.11 0.65 ± 0.03 0.029 ± 0.009
28 30/25°C 8.71 ± 1.34

**
5.68 ± 0.89

**
2.13 ± 0.51

**
0.91 ± 0.04 0.112 ± 0.016

**20/15°C 5.61 ± 0.69 3.64 ± 0.54 1.18 ± 0.18 0.79 ± 0.08 0.078 ± 0.018
42 30/25°C 15.94 ± 2.26 10.46 ± 1.63 4.06 ± 0.73 1.41 ± 0.15 0.223 ± 0.033

20/15°C 14.56 ± 1.77 9.11 ± 1.30 3.71 ± 0.53 1.74 ± 0.19 0.144 ± 0.016

Data are mean ± standard deviation (n = 17 at day 0, n = 11–12 between day 14 and day 28 and n = 5 at day 42). *P < 0.05; **P < 0.01.

Figure 2 Changes in relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf weight ratio (LWR) and
specific leaf area (SLA) for successive 14-day periods after transfer to two different day/night temperatures of 20/15°C (�) and 30/
25°C (�). Each point represents the mean ± standard deviation of the measurements on the different plants. For the 20/15°C (LT)
plants, n = 11 between days 0 and 15, n = 11 between days 14 and 28 and n = 5 between days 28 and 42. For the 30/25°C (HT)
plants, n = 12 between days 0 and 15, n = 12 between days 14 and 28 and n = 5 between days 28 and 42. *P < 0.05; **P < 0.01.

Temperature and photosynthesis in rose shoots 257

© 2008 Japanese Society of Soil Science and Plant Nutrition

dry weight increment per dry weight per day, was
higher in the HT plants than in the LT plants during the
first 14 days, but this difference became smaller between
day 14 and day 28, and was reversed between day 28
and day 42 (i.e. was higher in the LT plants). The
change in NAR was similar to that of RGR, and NAR
between day 28 and day 42 was also higher in the LT
plants. The LAR was greater in the HT plants throughout
the period. This was caused by greater LWR and SLA in
the HT plants, although the difference between day 14
and day 28 was not significant.

DISCUSSION

Our results with rose assimilation shoots indicate that
low growth temperature (20/15°C) initially suppressed
the photosynthetic rate during the first 7 days, but pro-
longed growth at low temperature enhanced potential
photosynthesis (Fig. 1, Tables 1,2). Similar trends have
been reported for several cold-habitat plants, such
as spinach (Holaday et al. 1992), Arabidopsis (Strand
et al. 1999), winter rye (Hurry et al. 1994), winter
wheat and winter rape (Hurry et al. 1995). In these
plants, low temperature increases the activities of
several photosynthetic enzymes, such as Rubisco, stromal
fructose-1,6-bisphosphatase and sucrose-phosphate
synthase. Yamori et al. (2005) also reported that the
amount of Rubisco increased in spinach leaves grown
under low temperature. In addition, Sage and Kubien
(2007) have recently pointed out that plants acclimated
to cooler temperatures often exhibit enhanced Rubisco
content. As shown in Table 2, as low temperature
enhanced total leaf-N content, such increases in the
photosynthetic components may have been associated
with an increase in leaf-N content at low temperature.
In our case, however, the photosynthetic rate at growth
temperature was lower in the LT plants just after
transfer to the low temperature. According to Kim and
Lieth (2003), the photosynthetic rate in rose does not
differ between 20 and 30°C. Therefore, an increase in the
amount of Rubisco as well as in the photosynthetic
capacity during growth at low temperatures can be one
of acclimation phenomena to low temperatures in rose.
Actually, in rice, which belongs to a typical summer
crop, the photosynthetic rate continuously decreased
during growth at 20/17°C and was never restored
(Hirotsu et al. 2004).

Rubisco is a limiting factor for light-saturated photo-
synthesis under atmospheric CO2 levels (Evans 1986;
Makino et al. 1985). However, although Rubisco content
at day 42 was 2.8-fold greater in the LT plants (Table 2),
the photosynthetic rate at 25°C was only 1.6-fold higher
(Table 1). Similarly, total leaf N and Chl contents were
1.8-fold and 1.7-fold greater in the LT plants than in

the HT plants. Thus, the increase in Rubisco content
during the growth at low temperature did not quantita-
tively lead to an increase in potential photosynthesis.
The reason for this discrepancy is not known, but one
possible explanation is an increase in the resistance to
CO2 diffusion from the intercellular airspace to the
chloroplasts in the low-temperature-grown rose. Makino
et al. (1994) suggested the possibility that the conduct-
ance to CO2 diffusion between the intercellular airspace
and the chloroplasts decreases when rice is grown under
low temperature. However, although Makino et al. (1994)
observed a large decrease in stomatal conductance during
growth at low temperature, stomatal conductance in
rose increased (Table 1). Thus, an increase in the stomatal
conductance at low growth temperatures may also be
one of acclimation phenomena to low temperatures in
rose. Another possibility is that Pi regeneration limitation
occurred in the LT plants. A selective enhancement of
Rubisco content often leads to a photosynthetic limitation
in Pi regeneration (Makino and Sage 2007).

The RGR was higher in the HT plants during the first
28 days and LAR was higher in the HT plants throughout
the experimental period (Fig. 2). Total leaf area was
also always greater in the HT plants (Table 3). These
results indicate that it can take less time to obtain
appropriate leaf area of the bent shoots in 30/25°C
cultivation than in 20/15°C cultivation (Fig. 2, Table 3).
In fact, the expansion rate of the leaf was faster in the
HT plants (data not shown). Thus, HT led to a rapid
leaf expansion and resulted in a higher initial growth
rate. However, because potential photosynthesis was
not enhanced, we conclude that HT does not suit the
growth of the assimilation shoots of rose before shoot
bending. In sweet pepper (Nilwik 1981) and Secale cerea
(Huner 1985), low temperature also led to decreases in
the LAR and the SLA. In these plants, narrower and
thicker leaf development was observed. Such morpho-
logical characteristics were similar to those found for
rose (Fig. 2).

Loveys (2002) reported that there is a species-dependent
difference in temperature response of NAR and it deter-
mines a difference in RGR under different temperatures.
In rose, NAR increased during growth at low temperatures
(Fig. 2), and this increase led to a large increase in RGR
during the late growth. In addition, the increase in NAR
compensated for a decrease in LAR over the whole
growth period. This increase in NAR may have been
caused by an increase in the amount of Rubisco during
growth at the low temperature (Table 2).

Conclusions
Our results clearly indicate that the photosynthetic capacity
in rose strongly depends on the growth temperature even
if the nutrition conditions are the same. Photosynthetic

258 A. Ushio et al.

© 2008 Japanese Society of Soil Science and Plant Nutrition

capacity is initially suppressed under growth at 20/
15°C, but prolonged growth at a low temperature
enhances potential photosynthesis. This is associated
with increases in Rubisco and N contents in a leaf. In
addition, enhanced photosynthesis leads to increases in
NAR and RGR at the level of the whole plant. Thus, to
enhance the photosynthetic capacity in rose assimilation
shoots, cultivation at 20/15°C is better than cultivation
at 30/25°C.

ACKNOWLEDGMENTS

We thank Mr Hideo Shimaji for his valuable comments
and support over the period of this research.

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