**Saffron**

*Crocus sativus* L., a perennial flowering plant and most expensive spice in the world. The economic part is the dried stigmas which are the source of a natural dye. It is mainly used as a colouring agent, for dyeing silk and fabric rugs, painting, cooking and as medicine. The Colouring principle: Crocin (4–6%) isolated in the crystalline state by Karrer et al. [19].


**7. Appendix**

*Mastery-development goals (5 items; Cronbach Alpha value=.75)*

*Performance-approach goals (2 items; Cronbach Alpha value=.65)*

*Extrinsic work goals (3 items; Cronbach Alpha value=.77)*

*Social enhancement goals (4 items; Cronbach Alpha value=.66)*

#### *Deep strategies (4 items; Cronbach Alpha value=.70)*

248 International Perspectives of Distance Learning in Higher Education

Future studies need to examine the effects of these important motivational variables on distance learners' achievement level. In particular, it will be interesting to examine if

Understanding distance learners' motivation will have practical implications for designing an engaging learning environment to support distance learners. In particular, the current findings suggest that building a distance learning system that promotes a mastery focus, encourage high performance, addresses personal development needs, and reinforces

Ames (1992) have discussed various classroom dimensions that promote mastery and engagement, such as the nature of task, authority, evaluation, and recognition. Using this perspective, distance educators can engage in a self-reflective process and ask critical questions, such as: Will distance learners find the learning units, activities and assigned readings interesting, challenging and helpful in developing their self-efficacy? Have the course and assessment items designed in such a way that distance learners are given abundant opportunities to develop independence, autonomy and choice? Is the learner support system effective in assisting learners to regulate their learning and progress? Are timely feedback on their progress and performance in the course provided to distance

These are important questions for distance educators who intend to create a motivating and engaging distance learning environment. Certainly, more research is required to look into the nature of distance learners' motivation in order to inform distance educators to make evidence-informed decisions on these critical questions. The current study has contributed to this effort by researching distance learners' motivation using an achievement goal

efficacy and control beliefs are fundamental to motivating distance learners to learn.

extrinsic work goals will be associated with a high level of achievement.

*Mastery-development goals (5 items; Cronbach Alpha value=.75)*

*Performance-approach goals (2 items; Cronbach Alpha value=.65)*

I take this course because it helps my career advancement.

*Social enhancement goals (4 items; Cronbach Alpha value=.66)*

I want to get a good result in this course.

*Extrinsic work goals (3 items; Cronbach Alpha value=.77)*

In this course, I would like to show that I am more capable than other students.

I take this course because I want to gain the related professional qualification.

Acquiring the knowledge in this course will enable me to help others and contribute to

I believe that my parents will be honoured when I do this course and its associated

 I do in this course because I want to learn something new. I found a sense of personal satisfaction in doing this course.

learners?

perspective.

**7. Appendix**

the society.

degree programme.

#### *Surface strategies (2 items; Cronbach Alpha value=.66)*

#### *Self-monitoring strategies (8 items; Cronbach Alpha value=.77)*

#### *Time Management (2 items; Cronbach Alpha value=.68)*

#### *Effort Management (2 items; Cronbach Alpha value=.61)*

#### *Help seeking (2 items; Cronbach Alpha value=.76)*

#### *Learning attitudes (7 items; Cronbach Alpha value=.83)*

#### *Efficacy Beliefs (3 items; Cronbach Alpha value=.72)*

#### *Control beliefs (3 items; Cronbach Alpha value=.65)*

The Role of Self-Efficacy, Control Beliefs and

3514

0268-0513

0007-1013

1996), pp. 181-192, ISSN 0361-476X

No.1, pp. 1–21, ISSN 0046-1520

pp.316-330, ISSN 0022-0663

22, No.4 (Oct 1997), pp. 415-435, ISSN 0361-476X

Vol.80, No.4, (Dec 1988), pp. 514-523, ISSN 0022-0663

*Learning*, Vol.21, No.1, pp. 33-48, ISSN 0268-0513

1998), pp. 221-230, ISSN 0305764X

Achievement Goals on Learning Among Distance Learners 251

Greene, B. A., & Miller, R. B. (1996). Influences on achievement: Goals, perceived ability, and

Grant, H., & Dweck, C. S. (2003). Clarifying achievement goals and their impact*. Journal of*

Harackiewicz, J. M., Barron, K. E. & Elliot, A. J. (1998) Rethinking achievement goals: When

Harackiewicz, J. M., Barron, K. E., Tauer, J. M., Carter, S. M., & Elliot, A. J. (2000). Short-term

Hurd, S. (2006). Towards a better understanding of the dynamic role of the distance

approaches. *Distance Education*, Vol.27, No.3, pp. 303-329, ISSN 0158-7919 Kaplan, A., & Midgley, C. (1997). The effect of achievement goals: Does level of perceived

Lyall, R., & McNamara, S. (2000). Influences on the orientations to learning of distance

Meece, J. L., Blumenfeld, P. C., & Hoyle, R. H. (1988). Students' goal orientations and

Miller, C., & Smith, C. (1998). Professional development by distance education: Does

Miller, R. B., Behrens, J. T., Greene, B. A., & Newman, D. (1993). Goals and perceived ability:

Ng, C. H. (2006). The role of achievement goals in the completion of a course assignment:

Ng, C. H. (2008). Multiple goals learners and their differential patterns of learning.

Ng, C. H. (2009). Profiling learners' achievement goals in completing academic essays.

Nolen, S. B. (1988). Reasons for studying: Motivational orientations and study strategies.

Pintrich, P. R. (1989). The dynamic interplay of student motivation and cognition in the

college classroom, In: *Advances in motivation and achievement*, Vol.6, M. L. Maehr & P. R. Pintrich (Eds.), pp. (117-160), JAI Press, ISSN 0749-7423, Greenwich CT.

*Educational Psychology*, Vol.28, No.4, pp. 439-456, ISSN 0144-3410

*Educational Psychology*, Vol.29, No.3, pp. 279-296, ISSN 0144-3410

*Cognition and Instruction*, Vol.5, No.4, pp. 269-287, ISSN 0737-0008

*Educational Psychology*, Vol.18, No.1 (Jan 1993), pp. 2-14, ISSN 0361-476X Morgan, A. R., Taylor, E., & Gibbs, G. (1982). Variations in students' approaches to studying.

cognitive engagement. *Contemporary Educational Psychology*, Vol.21, No.2, (April

*Personality and Social Psychology*, Vol. 85, No.3, (Sept 2003), pp.541-553, ISSN 0022-

are they adaptive for college students and why? *Educational Psychologist*, Vol.33,

and long-term consequences of achievement goals: Predicting interest and performance over time. *Journal of Educational Psychology*, Vol.92, No.2, (June 2000),

language learner: learner perceptions of personality, motivation, roles, and

academic competence make a difference? *Contemporary Educational Psychology*, Vol.

education students in Australia. *Open Learning*, Vol.15, No.2, pp. 107-121, ISSN

cognitive engagement in classroom activities. *Journal of Educational Psychology*,

distance lend enhancement? *Cambridge Journal of Education*, Vol.28, No.2, (June

Impact on student valuing, self-regulation, and persistence. *Contemporary*

*British Journal of Educational Technology*, Vo.13, No.2, (May 1982), pp.107-113, ISSN

Examining the effects of performance-approach goals and combined goals. *Open*

#### **8. References**

Aiken, L. S., & West, S. G. (1991). *Multiple regression: Testing and interpreting interactions*, Sage

Ames, C. (1992). Achievement goals and the classroom motivational climate. In: *Student*

Ames, C., & Archer, J. (1988). Achievement goals in the classroom: Students' learning

Barron, K. E. & Harackiewicz, J. M. (2001) Achievement goals and optimal motivation:

Biggs, J. (1987). *Student approaches to learning and studying*, Australian Council for

Bouffard, T., Vezeau, C., & Bordeleau, L. (1998). A developmental study of relation between

Braten, I., Samuelstuen, M. S., & Stromso, H. I. (2004). Do students' self-efficacy beliefs

Cochrane, C. (2000). The reflections of a distance learner 1977-1997. *Open Learning*, Vol.15,

Darnon, C., Harackiewicz, J. M., Butera, F., Mugny, G., & Quamzade, A. (2007). Performance-

Elliot, A. J. (1997) Integrating the 'classic' and 'contemporary' approaches to achievement

Elliott, E. S., & Dweck, C. S. (1988). Goals: An approach to motivation and achievement.

Elliot, A., & Harackiewicz, J. (1996). Approach and avoidance achievement goals and

Eppler, m. A. & Harju, B. L. (1997) Achievement motivation goals in relation to academic

*Psychology*, Vol.70, No.3, (March 1996), pp. 968-980, ISSN 0022-3514

*perceptions in classroom*, D. H. Schunk & J. L. Meece (Eds.), pp. (327-348), Lawrence

strategies and motivational processes. *Journal of Educational Psychology*, Vol.80,

testing multiple goal models, *Journal of Educational Psychology*, Vol.80, No.5, pp.

combined learning and performance goals and students' self-regulated learning. *British Journal of Educational Psychology*, Vol.68, No.3, (Sept 1998), pp.309-319, ISSN

moderate the effects of performance goals on self-regulatory strategy use? *Educational Psychology*, Vol.24, No.2, (April 2004), pp.231-247, ISSN 0144-3410 Cannon, M. M., Umble, K. E., Stechler, A., & Shay, S. (2001). "We're living what we're

learning": Student perspectives in distance learning degree and certificate programs in public health. *Journal of Public Management Practice*, Vol.7, No.1, pp. 49-

approach and performance-avoidance goals: When uncertainty makes a difference. *Personality and Social Psychology Bulletin*, Vol.33, No.6, pp. 813-827, ISSN 0146-1672 Dearnley, C., & Matthew, B. (2000). A group of nurses experience open learning: Exploring the impact. *Open Learning*, Vol.15, No.2, pp. 191-206, ISSN 0268-0513 Dweck, C. S. (1986). Motivational processes affecting learning. *American Psychologist*, Vol.41,

motivation: a hierarchical model of approach and avoidance achievement motivation, In: *Advances in motivation and achievement*, Vol. 10, M. L. Maehr & P. R.

*Journal of Personality and Social Psychology*, Vol.54, No.1, (Jan 1988), pp. 5-12, ISSN

intrinsic motivation: A mediational analysis. *Journal of Personality and Social*

performance in traditional and non-traditional college students, *Research in Higher*

Publications, ISBN<PHONE_NUMBER>, Newbury Park, C.A.

Educational Research, ISBN<PHONE_NUMBER>, Melbourne.

Erlbaum, ISBN 978-0-8058-0982-4, Hillsdale, NJ.

No.3, pp. 260-267, ISSN 0022-0663

706–722, ISSN 0022-0663

0007-0998

0022-3514

59, ISSN 1078-4659

No. 1, pp. 17-34, ISSN 0268-0513

No.10, pp. 1040-1048, ISSN 0003-066X

Pintrich (Eds), ISBN, (Greenwich, JAI Press Inc).

*Education*, Vol.38, No.5, pp. 557–573, ISSN 0361-0365

**8. References**

**12**

Ingrid Helleve *University of Bergen*

*Norway*

**Differences and Similarities in Approach**

**Between Classroom and Distance Learning**

This chapter will focus on differences and similarities between classroom and distance learning. What should count as learning and knowledge when information is available for everybody all the time? What are the consequences of these questions for teachers? These are questions that will be dealt with throughout the chapter. The aim of this Norwegian study is to single out what characterises productive interactions in ICT- (Information and communication technology) supported communities of learners, based on research from three different case studies. The study is based on the assumption that when teachers are designing and guiding learning communities there are some common features across agegroups and learning environments. Common for the three communities is that educational technology is supposed to serve as a space for collaborative writing activities. Across classrooms and distance learning there are some basic differences and similarities that will be discussed and illustrated through three different studies carried out between pupils in a classroom, on-campus students and distance learning students. The first study is carried out in 2nd grade in primary school where the students were supposed to write common texts by means of stand-alone-computers in the class-room. The next study deals with the experiences of 10 campus students in a blended environment. The students met every day, but were also supposed to collaborate online. The third study deals with distance learning. A group of five students called themselves the "*magic group*." They were student teachers who were supposed to publish portfolios and give feedback to each other. The research methods that are used are observations of the activities in the classroom, interviews and analysis of written texts. The conversation taking place when the pupils were writing common texts by means of the computers were recorded and analysed. The written online material is based on portfolios, feedback processes and online discussions. Further pupils, students and teachers in all three studies are interviewed. The aim of this chapter is to look across the borders of distance- and classroom learning in search of differences and

Schools as we have known them for hundreds of years have gathered people for the purpose of learning. Educational institutions are organized social societies. Currently it is relevant to raise the question of how to legitimize organized teaching and learning in a

**1. Introduction**

similarities.

**2. Why organized teaching and learning?**


The normality and homogeneity of variance for all the variables were determined based on Shapiro–Wilk's test (at a significance level of α = 0.001) and Levene's test (at a significance level of α = 0.05). Grubbs' test was used to detect and remove single outliers in the experimental dataset. Two-way ANOVA analysis comparing the effects of taxon, salt treatment, and the interactions between them was used to assess differences between *P. pyraster* and *T. cordata* seedlings grown under salt treatment. The multiple comparison of means was performed using the Tukey honest significant difference (HSD) test (at significance levels of α = 0.05). Regression analysis was applied for assessment of the relationships between RWC, Ψwl, and the cumulative salt uptake.

Statgraphics Centurion XVII software (StatPoint Technologies, Warrenton, VA, USA, XVIII, license number: B480-E10A-00EA-P00S-60PO) was used for statistical data analysis.

#### **3. Results**

#### *3.1. Effect of Salinity on Growth and Mass Accumulation of Plant Organs*

*T. cordata* is more efficient in biomass production compared to *P. pyraster*, as per the documented data on the fresh weight (FW) and relative biomass increment (RBI) for the control plants (Table 2). Salinity (salt stress) had a strong but variable effect on the growth and mass accumulation of plant organs. RBI was significantly reduced for both species, but only for *T. cordata* was the decrease in both total FW and DW considered significant. Under salt treatment, the dry mass values of the aboveground organs (DWS) (−26%) as well as of the root (DWR) (−46%) were significantly reduced for *T. cordata* seedlings. The stem increment and leaf dry mass (DWL) were not affected, but salinity reduced the radial growth of the stem manifested by the decrease in the stem dry mass (SDW) (−35%) compared to the control (Table 2). The root growth in length (−47%) and volume (−54%) were also reduced. Under salt treatment, DWS and DWR were not significantly reduced for *P. pyraster* seedlings. *P. pyraster* adapted to salinity by investing in root growth, as it increased the number of root tips (NRT) and maintained a balanced root length. The average root diameter (ARD) and root volume (RV) (−32%) of the salt-treated seedlings were reduced. Salinity did not significantly change the R:S for *P. pyraster* seedlings, but this was reduced for *T. cordata*. Under salt treatment, *T. cordata* has limited root growth and preferentially accumulated dry mass in the aboveground organs (leaves). The substrate salinity negatively affected the growth and development of the leaves of both studied species, which is documented by the reduction in leaf area (LA) (*T. cordata* −26%, *P. pyraster* −35%) compared to control plants. In both species, the specific leaf area (SLA) was also reduced in response to salinity (Table 2). However, leaf injury symptoms only appeared in *Tilia* seedlings. The first signs of damage appeared after 35 days of the experiment (when 265 mL of 100 mM NaCl had already been applied to the substrate) in the form of leaf yellowing (Figure 2A) and, later, leaf edge burning (Figure 2B) and early leaf dropping (Figure 2B,C). *P. pyraster* seedlings showed no symptoms of leaf damage during the experiment.

#### *3.2. Leaf Water Status under Salt Treatment*

The water potential of the leaf tissues was influenced by the taxon as well as by the cumulative amount of applied saline solution at a concentration of 100 mM NaCl, which was regularly applied to the substrate. At the beginning of the experiment, the water potential of the leaf tissues was significantly higher for *T. cordata* (−0.71 MPa) compared to *P. pyraster* (−1.46 MPa). From the beginning of the salt treatment, *Tilia* maintained balanced Ψwl values (−1.00 ± 0.36) for 30 days, when 225 mL of 100 mM NaCl per plant had been added to the substrate (Figure 3). However, Ψwl was significantly reduced at the 40th day (−2.45 MPa) and 50th day (−3.22 MPa) of the salt treatment. *P. pyraster* maintained a balanced Ψwl (−1.50 ± 0.22 MPa) throughout the whole experiment (Figure 3). A significant increase in the water potential was observed only at day 30 of the experiment.

**Table 2.** The growth parameters and biomass allocation of *P. pyraster* and *T. cordata* seedlings in the pot experiment after 50 days of salt treatment. The multiple comparison of means (*n* = 20) was performed using the 95% Tukey honest significant difference (HSD) test. Data are the mean values and standard deviations (±SD). Mean values followed by different letters are significantly different.

T—taxon; S—salt treatment; T × S—interaction between the taxon and salt treatment; LA—leaf area; SLA—specific leaf area; DWL—dry mass of leaves; SDW—dry mass of the stem; DWS—dry mass of shoots; RBI—relative biomass increment; FWS—fresh mass of shoots; FWR—fresh mass of roots; RL—root length; SRL—specific root length; DWR—dry mass of roots; RSA—root surface area; ARD—average root diameter; NRT—number of root tips; RV—root volume; R:S—root to shoot mass ratio.

**Figure 2.** The leaf damage on *T. cordata* seedlings due to a high concentration of Na+ ions. The first symptoms appeared after 35 days of salt treatment in the form of (**A**) leaf yellowing; (**B**) leaf edge burning; and (**C**) early leaf fall after 50 days of salt treatment.

RWC values for *P. pyraster* and *T. cordata* seedlings demonstrated differences in the species responses to salinity (Figure 4). At the beginning of the experiment, the RWC values were in the range of 96.95 ± 1.34% for *T. cordata* and in the range of 96.19 ± 0.26% for *P. pyraster*. On day 30 of the salt treatment, a significant decrease in the RWC (84.04 ± 7.5%) was observed for *P. pyraster*, but the values of this parameter later increased and remained stable until the end of the experiment (89.48 ± 4.80%). The RWC values for *T. cordata* steadily declined from the beginning of the saline treatment (Figure 4), but a significant decrease (80.71 ± 6.49) was demonstrated on day 50 of the experiment after the cumulative NaCl intake per plant exceeded 300 mL.

**Figure 3.** Box plot for the water potential of leaf tissues (Ψwl) of the seedlings of *P. pyraster* and *T. cordata* measured at the beginning of the experiment (0 mL) and during the experiment when treated with 100 mM NaCl solution. Significant differences between measurements (*p* < 0.05) are indicated by different letters.

**Figure 4.** Box plot for the relative water content (RWC) for seedlings of *P. pyraster* and *T. cordata* measured at the beginning of the experiment (0 mL) and during the experiment when treated with 100 mM NaCl solution. Significant differences between measurements (*p* < 0.05) are indicated by different letters.

The obtained data document different reactions of the studied species to salinity and, therefore, varying mechanisms for regulating water uptake. The leaf tissues of *T. cordata* have a demonstrably higher water potential than *P. pyraster*; however, at a higher cumulative salinity level (above 300 mL per plant), the values of Ψwl decreased sharply, similar to the RWC. *P. pyraster* maintained a balanced water potential and relative water content in the leaves under salt treatment, even at a higher cumulative NaCl intake per plant.

#### *3.3. Salt Ion Uptake and Distribution*

Significant differences were observed in the studied species in terms of the uptake and distribution of salt ions to the plant organs. During the experiment, the same amount of salt solution was applied to the substrate per plant, but *T. cordata* seedlings absorbed significantly more Na+ ions compared to *P. pyraster* (Figure 5). *P. pyraster* accumulated Na+ ions mainly in the root (75%), and a low amount of Na+ was distributed to the aboveground organs—stems (19%) and leaves (6%) (Figure 6). Similarly, *T. cordata* retained the majority of Na+ ions in the root system (55%); however, a relatively high proportion was also distributed to the stems (34%) and leaves (11%). Compared to the untreated control, *T. cordata* significantly increased the uptake of K+ ions, and preferentially distributed them to the stem and leaves under salt treatment. The K+ content did not change in the leaves of *P. pyraster* compared with the control, but it was significantly reduced in the stem and root system (Figure 4). Increased NaCl uptake competed with K+ intake and led to a significant change in the K+/Na+ ratio in the plant organs. The significant differences in the K+/Na+ ratio are influenced by the increased salt content in the substrate and are species-specific (Figure 5). In the roots of *P. pyraster*, the K+/Na+ ratio decreased (−81%) from 15.1 (control) to 2.8 (salt-treated seedlings). In *T. cordata* roots, the change in the K+/Na+ ratio was even more pronounced (−94%) and decreased from 17 (control) to 1 for salt-treated seedlings (Figure 5). A significant decrease (−96%) in the K+/Na+ ratio was detected in the leaves of *T. cordata* in comparison to both the control as well as salt-treated *P. pyraster* seedlings (−77%). Compared to *P. pyraster*, *T. cordata* distributed a six times higher amount of Na+ to the leaves, which negatively affected the condition of the leaf apparatus and induced the development of necrosis (Figure 2). *P. pyraster* blocked salt uptake at the level of the root system, and thus protected its leaves from intoxication and damage.

#### *3.4. Effect of Salinity on Photosynthetic Pigments*

The content of photosynthetic pigments for the seedlings of the studied tree species is species-specific. Compared to *T. cordata*, *P. pyraster* has a significantly higher content of photosynthetic pigments according to the relevant parameters for the control and salttreated plants (Table 3). A significant reduction in the chlorophyll content was observed in the leaves of *T. cordata* seedlings after the application of 100 mM NaCl in a cumulative volume of 365 mL NaCl per plant. The contents of total chlorophyll (Chl*ab*), Chl*a*, and Chl*b* were decreased by NaCl stress. The carotenoid content and the Chl *a/b* ratio did not change for the salt-treated seedlings compared to the control. *P. pyraster* seedlings maintained a balanced content of photosynthetic pigments in the leaves during 50 days of the continual salt treatment.

**Table 3.** The contents of photosynthetic pigments in the leaves of *P. pyraster* and *T. cordata* seedlings after 50 days of salt treatment. Mean values followed by different letters are significantly different.

T—taxon; S—salt treatment; T × S—interaction between the taxon and salt treatment.

**Figure 5.** The salt ion content (Na+, K+, and K+/Na+) in the (**A**) leaves, (**B**) stems, and (**C**) roots of two-year-old seedlings of *P. pyraster* and *T. cordata* after 50 days of continuous 100 mM NaCl saline application and in the control. The bars indicate the mean values of the ion content in the plant organs from a sample of 10 plants. Significant differences (*p* < 0.05) are indicated by different letters.

**Figure 6.** The content and distribution of Na+ ions in the plant tissues (leaves, stem, roots) of *P. pyraster* and *T. cordata* seedlings grown for 50 days under salt treatment with 100 mM NaCl solution. The values above the bars indicate distribution to the plant organs as a percentage of the total Na+ ion uptake.

#### *3.5. Effect of Salinity on Leaf Gas Exchange*

Species–specific differences were observed in all examined gas exchange parameters (gs, E, An, WUE) (Table 4). The *P. pyraster* seedlings had significantly reduced gs and E in response to salinity on day 20 of regular NaCl application to the substrate. The reductions in gs and E were observed in response to the osmotic phase of the salinity stress. During the experiment, *P. pyraster* seedlings maintained balanced values of both parameters (gs, E) compared to the control under increasing cumulative NaCl intake to the substrate. *P. pyraster* seedlings had balanced values of An and WUE without significant changes compared to the control throughout the whole experiment. In response to the salt treatment, the seedlings of *T. cordata* had reduced gs and increased WUE on days 20 and 40 of the experiment, when the cumulative salt uptake was 155 and 295 mL per plant, respectively. The transpiration rate decreased significantly on day 40 of the experiment (0.27 ± 0.12 mmol H2O m−2·s−1) compared to the control (0.58 ± 0.19 mmol H2O m−2·s−1). A significant decrease in An was observed on day 50 of the experiment, which indicates that salt stress has a negative impacts on photosynthesis. The decreases in the stomatal conductance and transpiration rate were transient for *P. pyraster* seedlings, which maintained balanced photosynthesis even under conditions of higher cumulative salt content in the substrate.

**Table 4.** The parameters of leaf gas exchange in the leaves of *P. pyraster* and *T. cordata* seedlings after 50 days of salt treatment (*n* = 5). Mean values followed by different letters are significantly different.

DOT—day of treatment; CSI—cumulative NaCl intake per plant; T—taxon; S—salt treatment; T × S—interaction between the taxon and salt treatment; An—net assimilation rate; E—transpiration rate; gs—stomatal conductance to water vapor; WUE—water use efficiency.

#### **4. Discussion**

Salinity causes severe physiological dysfunctions in plants in addition to indirect damage, even at low concentrations [25]. The growth reduction due to salinity has been documented for many plant species, but not halophytes [26]. Several authors particularly refer to the reduction in shoot growth [2,36–39]. In our study, salinity significantly reduced the fresh and dry matter of *T. cordata* seedlings (FW: 56%; DW: 34%). A significant reduction in shoot and root matter was not confirmed for *P. pyraster* seedlings under salt treatment, where the mass accumulation did not change compared to the control plants. *P. pyraster* invested in root growth, increased the number of root tips and maintained the balanced parameters RL and SRL under salt treatment. Our previous research shows that *P. pyraster* maintains a balanced ratio of the mass distribution between underground and aboveground organs (R:S) even under conditions of water scarcity [40,41]. The findings suggest the effective water uptake and retention of salt ions in the root system of *P. pyraster*. *T. cordata* demonstrably reduced the length of the root (−47%) and the number of root tips (−47%) under salt treatment, which reduced the exploratory capacity of the root system. The obtained data indicate both effective water uptake as well as retention of salt ions in the root system for *P. pyraster*. The continuous growth of roots allows for the continuous exploration

of new soil for water and the partial alleviation of water stress through improvements in the water supply [42]. Increasing the proportion of root system mass in plants exposed to salinity has a positive effect through the increased retention of toxic salt ions in the root, thus limiting their transfer to aboveground organs (especially leaves) [2], which can be a typical mechanism of resistance or survival in condition of excessive salinity [43,44].

Under salt treatment, the seedlings of the studied tree species had reduced leaf area (*Pyrus*: 35%; *Tilia*: 26%), which is considered a typical response to salt stress. *P. pyraster* formed smaller leaves and showed significantly reduced shoot growth (−43%). According to Munns and Tester [1], a significant reduction in the size of individual leaves or the number of shoots is the main effect of salinity that manifests in dicotyledonous species. *T. cordata* seedlings maintained balanced stem growth and dry matter accumulation in the leaves even after salt treatment. The reduction in the leaf area was due to damage and early leaf fall. The first symptoms of leaf damage appeared on *T. cordata* seedlings on day 35 from the beginning of the salt treatment. The salt ions were accumulated in the leaves to the level of toxic concentration, which caused leaf damage and the decay of photosynthetic pigments. According to Munns and Tester [1], the "ion-specific" phase of plant response to salinity begins when a toxic level of salt concentration in leaf tissues is reached. According to these authors, the leaf development during long-term salinity is mainly influenced by the ability of plants to prevent the accumulation of salt ions in assimilation organs in addition to producing new leaves faster than salt-intoxicated leaves are dying.

The control of salt uptake by the roots and the regulation of salt distribution to the aboveground organs are important mechanisms in preventing the concentration of salt ions in plant leaves. It is considered to be a significant characteristic of the plant tolerance to salinity [45–49]. Both studied tree species accumulated salt ions mainly in the root (*Pyrus* 75% and *Tilia* 55%). However, when the same cumulative volume of the salt solution was applied to the substrate (365 mL) in each plant, *T. cordata* seedlings deposited 4.5 times more Na+ ions in the plant tissues (Figure 5) and specifically distributed six times more Na+ ions to the leaves compared to *P. pyraster*. A high salt uptake negatively affected the condition and functionality of the assimilation apparatus of *T. cordata*. *P. pyraster* blocked the salt uptake in the root system (75%) and deposited a portion of the Na+ ions the stem (19%), thus protecting the leaf apparatus from intoxication and damage. The effective limitation of the salt uptake and the deposition of ingested salt ions in the root can be considered a manifestation of the tolerance of *P. pyraster* seedlings to salinity. The mechanisms for blocking salt ions in the stem and root system have been identified for *P. amygdaliformis* and *P. elaeagrifolia* [50]. These species showed no symptoms of leaf damage even after 30 days of 150 mM NaCl treatment nor any changes in the photosynthetic rates. A similar restriction and blocking of Na+ ions in trunk wood is reported for *P. communis* by Boland et al. [51].

Salinity affects nutrient uptake and significantly reduces K+ content in the plant tissues of many species [52–54]. The exclusion of Na+ from plant tissues is considered a mechanism for the optimization of the K+/Na+ ratio [55,56]. The ability of plants to optimize the K+/Na+ ratio (through K+ retention or the prevention of Na+ accumulation in leaves) is considered to be a key feature of salt tolerance [57,58]. Under salt treatment, *T. cordata* significantly increased the uptake and distribution of K+ to the leaves and stems, while *P. pyraster* maintained a balanced K+ content in only the leaves, probably due to the effective restriction of Na+ transfer and deposition in leaves (Figure 5A,B). The seedlings of the studied tree species each applied different strategies for the optimization of the K+/Na+ ratio in the plant tissues, i.e., an increased K+ uptake (*T. cordata*) or restricted uptake and transfer of Na+ (*P. pyraster*).

Salinity also affects the water–plant relationship, as the salts present in the substrate prevent the absorption of water by the root system due to the osmotic effect [59]. The accumulation of salts in the root zone causes a decrease in the osmotic potential, with a consequent decrease in the water potential [60–62]. Under salinity conditions, plants show symptoms of dehydration, indicating a lower water potential due to poor water uptake or less water availability in the substrate [63,64]. *P. pyraster* quickly reduced water uptake due to the higher concentration of the salt ions in the root zone. The decrease in the RWC (Figure 4) was the result of a high concentration of the external salt solution, which according to Greenway and Munns [65], causes osmotic stress and dehydration at the cellular level. *P. pyraster* was effective in the restriction of salt uptake and transport to the leaves. Salt ions were maintained in the root system and in the stem, which allowed the restoration of the water uptake through a slight increase in the water potential of the leaf tissues (Figure 3). The balanced values of the water potential were maintained for *P. pyraster* even under increasing cumulative salt content in the substrate. The decrease in the RWC and Ψwl under increasing salinity was observed for *T. cordata* seedlings. The significant decrease in Ψwl (−2.48 MPa) was accompanied by the severe symptoms of the leaf damage. *T. cordata* was not effective in blocking salt uptake by the root system and transported Na+ ions to the leaves that were damaged due to salt accumulation. The *T. cordata* seedlings were particularly affected by ionic stress, which dominates only at high levels of salinity or in sensitive species that lack the ability to control Na+ transport [1].

The data obtained in our study indicate the species-specific content of photosynthetic pigments in the leaves of *T. cordata* and *P. pyraster*. The contents of Chl*a*, Chl*b*, and Chl*ab* were significantly reduced only for *T. cordata* seedlings after the regular application of 100 mM NaCl solution in the total cumulative volume of 365 mL per plant. The chlorophyll parameters were not reduced for *P. pyraster* compared to the control plants. The balanced content of photosynthetic pigments indicates the tolerance of *P. pyraster* to salinity. Salinetolerant plants have increased or unchanged chlorophyll content in the leaves when treated with salt, while chlorophyll content decreases in saline-sensitive plants [66,67]. Chlorophyll content in leaves is considered to be a biochemical indicator of the plant's tolerance to salinity [54]. Compared to *T. cordata*, *P. pyraster* seedlings have a significantly higher content of carotenoids, which, according to several studies, stabilize the photochemical processes of photosynthesis under stress conditions [68].

The seedlings of *P. pyraster* and *T. cordata* had reduced gs in the early response to salt treatment (after 20 days). The decrease in the transpiration rate (E) was observed only for *P. pyraster*. Stomatal plant responses are induced by the osmotic effect of salinity in the root zone [1]. A reduction in gs can prevent excessive water loss by transpiration, similarly to what has been observed for plants in desiccating soil [69]. *P. pyraster* limited water loss in the initial (osmotic) phase of salt stress and maintained a steady net photosynthetic rate (An) per unit area throughout the whole experiment. The rate of photosynthesis per leaf unit area often remains unchanged for salt-treated plants, even under reduced stomatal conductance [70]. It is associated with changes in the cell anatomy that give rise to smaller, thicker leaves and lead to higher chloroplast densities per unit leaf area [1]. As these authors state, when photosynthesis is expressed on the basis of a chlorophyll unit and not on the basis of leaf area, the reduction due to salinity can usually be measured. Therefore, the reduction in the leaf area due to salinity means that photosynthesis per plant is always reduced.

The ratio of stomatal opening and the level of photosynthesis (water use efficiency, WUE) are used as indicators of plant tolerance to osmotic stress [2,71]. *T. cordata* seedlings responded to salinity by increasing WUE. The increase in WUE was evident in two stages (on days 20 and 40) of the experiment, which is related to the increasing cumulative NaCl uptake per plant. Due to the high accumulation of Na+ ions in the leaves (ionic stress), the abovementioned adaptive reactions decreased the water potential of the leaf tissues and also significantly decreased the net photosynthetic rate (An) on day 50 of the experiment. Despite the adaptive reactions of *T. cordata* seedlings to osmotic stress, Ψwl significantly decreased on day 40 of the experiment due to the high accumulation of Na+ ions in the leaves (ionic stress). In the long term, a high salinity level had a negative impact on photosynthesis (decrease in An) in *T. cordata* seedlings. The accumulated salts in the leaves may inhibit enzymes involved in carbohydrate metabolism or exert a direct toxic effect on photosynthetic processes [1].

#### **5. Conclusions**

Differences in tolerance to salinity were identified for *P. pyraster* and *T. cordata* seedlings as well as different mechanisms when coping with salt stress. The results document the significance and role of the root system in the resistance and survival of woody plants under saline conditions. Balanced root growth improves water supply and has a positive effect via the retention of toxic salt ions, which restricts their transfer and accumulation in leaves.

Under salt treatment, *P. pyraster* maintained a balanced growth and biomass accumulation in the root, as observed in the balanced root parameters RL, SRL, FWR, and DWR, and the increased number of root tips. The *P. pyraster* seedlings reduced the size of the leaf area in response to osmotic stress, i.e., increased salinity in the root zone. The seedlings formed smaller leaves, effectively regulated stomatal conductance, and maintained balanced values of RWC and Ψwl. *P. pyraster* blocked salt uptake via the root system and in the stem, thus protecting the leaf apparatus from intoxication and damage. The rate of photosynthesis per leaf unit area (An) remained unchanged under increasing salinity throughout the whole experiment. The reduced water loss, effective limitation of the salt uptake, deposition of ingested salt ions in the stem and roots, balanced content of photosynthetic pigments, and protection of the leaf apparatus against intoxication observed in *P. pyraster* seedlings are indicative of their tolerance to salinity.

We consider the effective limitation of the salt uptake and deposition of the ingested salt ions in the root to be a manifestation of the tolerance of *P. pyraster* seedlings to salinity. This tree species reduced water loss in the initial (osmotic) phase of salt stress and maintained a steady rate of photosynthesis (An) per unit area throughout the whole experiment.

*T. cordata* seedlings regulated water loss (gs) and maintained balanced photosynthesis (An) in the early response to osmotic stress. However, they were not effective in the restriction of salt uptake by the root system. Na+ ions were transported to the leaves, which were damaged by the accumulation of salt ions. The seedlings were not able to cope with the ionic stress that dominates at high levels of salinity or in sensitive species that lack the ability to control Na+ transport. Despite adaptive reactions (gs regulation, growth restriction), a significant decrease in Ψwl and An was observed for salt-treated *T. cordata* seedlings. The obtained findings show weak tolerance to salinity for *T. cordata* in the juvenile stage of growth.

This comparison of the early stages of growth shows that *P. pyraster* can cope with salinity in urban conditions. However, a deeper study of the effects of different forms and levels of salinity on these tree species is necessary in order to further our understanding of the mechanisms of salinity tolerance.

**Author Contributions:** Conceptualization and design, V.P. and M.H.; investigation, M.H. and H.L.; methodology. V.P. and M.H.; resources, H.L.; formal analysis, V.P. and M.H.; validation, H.L.; writing the first draft, V.P. and M.H.; writing—review and editing, V.P. and M.H.; funding acquisition. V.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Kultúrna a Edukaˇcná Grantová Agentúra MŠVVaŠ SR (Cultural and Educational Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic) grant number 007SPU-4/2020. This publication was supported by the Operational Programme Integrated Infrastructure within the Sustainable Smart Farming Systems Taking into Account Future Challenges 313011W112 project. This study was co-financed by the European Regional Development Fund.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**

## *Article* **Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in** *Callistemon citrinus* **(Curtis) Plants**

**Stefania Toscano, Giovanni La Fornara and Daniela Romano \***

Department of Agriculture, Food and Environment (Di3A), Università degli Studi di Catania, Via Valdisavoia 5, 95123 Catania, Italy<EMAIL_ADDRESS>(S.T<EMAIL_ADDRESS>(G.L.F.) **\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The growth and aesthetic value of ornamental plant species used near coastlines are negatively influenced by salt spray. The presence of surfactants could enhance salt damage. To analyze the influences of salt spray and surfactants alone and in combination with each other, individual *Callistemon* plants were subjected to different treatments for 8 weeks: a solution simulating the composition of seawater (salt spray), a solution containing an anionic surfactant (surfactant), a solution with salt spray and anionic surfactant (salt plus surfactants), and deionized water (control). To study the influence of different climatic conditions, two growing periods, from January to March (I CP) and from May to July (II CP), were established. Salt spray, alone or with surfactant action, influences plants' growth and aesthetic features in different cycle periods. The percentage of leaf damage significantly increased with salt spray and salt plus surfactants during II CP (~27%). Additionally, the Na+ and Cl− contents were enhanced in the leaves in both CPs, but the contents in the roots were only enhanced in the II CP. The gas exchanges were significantly influenced by the treatments, especially during the II CP, when a reduction in net photosynthesis due to salt spray was observed starting from the second week of stress. At the end of the experiment, in both cycle periods, the leaf proline content increased in the salt spray and salt plus surfactants treatments. In both CPs, PCA revealed that the morphological and physiological parameters were directly associated with the control and surfactants treatments, whereas the mineral contents and biochemical parameters were directly correlated with the salt and salt plus surfactants treatments. The additive effect of surfactant stress, compared to salt stress, did not appear to be significant, with the exception of CP II, and for some parameters, the solubilization action of surfactants was favored by higher temperatures.

**Keywords:** coastal areas; ornamental plants; gas exchange; chlorophyll *a* fluorescence; enzyme activity; proline

#### **1. Introduction**

In coastal gardens and ornamental green areas, one of the main problems observed is related to salt stress originating from sea spray, which damages plants [1,2]. In this landscape, salt spray may limit plant growth and survival [3], altering coastal species' composition [4] and thus influencing the ornamental and ecological value of plant communities [5,6].

The negative effects of salts on ornamental plants are observed above all on their aesthetic appearance, which is an essential trait [7]. The visual aspect, although a subjective parameter, is of fundamental importance when evaluating the salt tolerance of landscape plants [8]. Among the different species used in urban landscaping, salt tolerance differs considerably [9] according to the morphological and physiological characteristics of different genotypes. Most ornamental plant species are non-halophytes; therefore, the assessment of salt tolerance is necessary [10]. The species selected for inclusion in urban green areas must not only survive but must also maintain suitable aesthetic characteristics [11].

**Citation:** Toscano, S.; La Fornara, G.; Romano, D. Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in *Callistemon citrinus* (Curtis) Plants. *Horticulturae* **2022**, *8*, 261. https://doi.org/10.3390/ horticulturae8030261

Academic Editor: Yanyou Wu

Received: 18 February 2022 Accepted: 16 March 2022 Published: 17 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Although plant species typical of coastal areas are resistant to the presence of salt on their leaves [12], plant growth and reproduction can be negatively affected when they are exposed to salt spray, and when salt water penetrates into the groundwater [13]. When salt is applied to roots, the damage is less significant than that seen from salt sprays [14]. Salt spray can reduce plant growth because it determines water stress; leads to the disruption of membranes and enzyme systems; causes the inhibition of nutrient uptake, necrosis, and the loss of leaves, and can lead to mortality [15].

In modern societies, surfactants are often used in the form of detergents and pesticides. Given their wide usage, these substances cause pollution in aquatic environments. The foliar absorption of sea salt through stomatal and cuticular penetration can be increased by the presence of surfactants in seawater [16].

The wide uses of surfactants in chemical industries, such as household products, industrial cleaning, ink, pharmaceuticals, and personal care, can affect the environment and human health. In 2014, 15.93 million tons of surfactants were used, and in 2022 this figure is expected to be over 1/3 more (24.19 million tons of surfactants) [17]. The phenomenon is worrying for the coastal ecosystem because about 10% of this amount is released into the sea [18].

The damage to coastal flora is mainly caused by the synergic effects of marine salt and surfactants, but also by the direct actions of the surfactants [19] that are responsible for the solubilization of cell membranes [20], the increase in the permeability of cuticles [21], and the dissolution of epicuticular waxes [22], all of which are processes that facilitate the foliar absorption of salt, and its phytotoxic effects. The effect of surfactants is to degenerate epicuticular waxes and modify the photosynthetic processes by causing the leaves to absorb greater quantities of sea salt [23].

Bussotti et al. [24] observed that resistance to salt spray depends on different behaviors determined by leaf structure (sclerophyllia, cuticle thickness). Reductions in gas exchanges and photosystem II (PSII) efficiency have been observed in the presence of salt spray because, although the cuticle of the leaves protects against external agents, it cannot prevent the penetration of ions and the consequent osmotic and ionic stress [25]. Modifications in the chlorophyll content and consequently reductions in photosynthesis activity occur in the presence of saline stress [26]. Stomatal limitation reduces the diffusion of CO2 in the mesophyll and can contribute to the generation of additional reactive oxygen species (ROS), which may cause irreversible damage [27].

Photosynthetic pigments can be damaged by ROS, altering the metabolism because they are highly reactive in the absence of protective mechanisms [28]. In cellular compartments, to counteract the effects of reactive oxygen species, plants have developed different mechanisms. Generally, plants activate various antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX)) [29]. In many species, a close relationship is clear between stress tolerance and antioxidant activity [30–32]. Catalase, guaiacol peroxidase, and L-ascorbate peroxidase are all involved in the plant's responses to biotic and abiotic stresses. In particular, these enzymes are involved in the scavenging of reactive oxygen species (ROS), partially reduced forms of atmospheric oxygen, that are highly reactive and capable of causing oxidative damage to the cell.

Among the ROS, the most dangerous is the superoxide anion; this is scavenged in plants by superoxide dismutase, which converts the superoxide anion to hydrogen peroxide [33]. Furthermore, in the context of the detoxification of toxic ROS, the superoxide anion is a fundamental component of the ascorbate-glutathione cycle [34].

In the presence of abiotic stress, plants accumulate metabolic components, which act as plant osmotic regulators, such as proline (Pro) and malondialdehyde (MDA) [35–37].

Few studies have been conducted on the response of plants to interactions between natural stresses in coastal areas; research has generally focused on single stress, leading to a suitable but incomplete understanding of the response mechanism of plants [38]. The effect of stress depends on both the intensity of the stress factor and on its duration and therefore makes it more difficult to understand the response mechanisms and interactions between other stresses [39]. In coastal areas, salt spray is often associated with pollutants, and the role of surfactants is critical in some cases of environmental damage to coastal vegetation [40]. Interactions with environmental conditions are often important as well; surfactant experiments carried out during winter produced damage lower down in the plants than experiments in warmer seasons [41].

To better understand the influence of some of the stronger factors affecting the ornamental plants used in the coastal landscape, we selected *Callistemon* of the Myrtaceae family; this entire genus is endemic of Australia and occurs in the form of shrubs or small trees. The *Callistemon* genus is characterized by a suitable tolerance to drought, salt, and root restriction stresses [42–44]. One of the most interesting ornamental species is *Callistemon citrinus* (Curtis) Skeels, characterized by fast growth and abundant blooming with bright colors and various shapes and volumes [45].

The research related to the effects of salt spray and its interactions with surfactants in ornamental shrubs is sparse. The aims of this study were to define (i) the influence of salt spray and surfactants and their interactions on *Callistemon* plants during two cycle periods with different climatic conditions; (ii) the morphological, physiological, and biochemical mechanisms involved in responding to salt spray and surfactants; (iii) the relationships among different parameters, in order to better understand the action mechanisms.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Treatments*

Both cycle periods were operated in an unheated greenhouse located near Catania, Italy (37◦41- N 15◦11- E 89 m a.s.l.). In 3 L pots (16 cm), filled with peat and perlite (2:1 *v*/*v*), rooted cutting (two months old) of *Callistemon* (*Callistemon citrinus* (Curtis) Skeels) were transplanted; 2 g L−1 of Osmocote Plus (14:13:13 N, P, K plus microelements) was added to each pot. A drip system with one 2 L h−1 emitter into each pot was used, and the plants were irrigated daily. The plants were subjected for 8 weeks to different treatments: (a) a solution simulating the composition of seawater-salt spray [46] with a concentration of 401.8 mM (salt spray); (b) a solution containing an anionic surfactant (sodium dodecylbenzene-sulfonate 82.52%, 50 mg L−1) (surfactants) [3]; (c) a solution containing salt spray and anionic surfactant (salt plus surfactants); (d) control plants treated only with deionized water (control). The plants were sprayed twice a week with different aqueous solutions. To avoid salt deposition on the soil, plastic discs were placed over the soil surfaces of the potted plants. To investigate the effects of different climatic conditions, two growing periods were considered: January to March (I CP), with transplant in November, and May to July (II CP), with transplant in February.

#### *2.2. Plant Materials and Treatments*

At the ends of both cycle periods, three pots per replication were randomly chosen for the determination of shoots' and roots' fresh and dry biomass, total leaf area, leaf number, chlorophyll content, and leaf damage. The dry biomass (DW) of the shoots (leaves plus stems) and the whole plant (leaves, stems, and roots) was determined by drying the weighed samples in a thermo-ventilated oven at 70 ◦C until constant weights were obtained. A leaf area meter (Delta-T Devices Ltd., Cambridge, U.K.) was used for determining the total leaf area and percentage of leaf damage (necrotic area on total area). The protocol of Moran and Porath [47] was adopted to measure the chlorophyll content. Dry leaves and roots were ground in a Wiley Mill and then passed through a 20-mesh screen for the mineral analysis. Na+ and Cl− contents were determined by chromatography on a Dionex IC 25 (Dionex Corp., Sunnyvale, CA, USA). Ion concentrations were expressed in g kg–1 DW.

#### *2.3. Chlorophyll Content, Gas Exchanges, Chlorophyll a Fluorescence and RWC Measurements*

The gas exchanges were registered during both cycle periods, every two weeks (10:00 am and 2:00 pm). Net photosynthetic rate (AN) and stomatal conductance (gs) were registered on fully expanded leaves through a CO2/H2O IRGA (LCi, ADC Bioscientific Ltd., Hoddesdon, UK). Simultaneously, the efficiency of PSII was registered using a modulated chlorophyll fluorimeter OS1-FL (Opti-Sciences Corporation, Tyngsboro, MA, USA). Using cuvettes, the leaves were darkened for 20 min before measurement. The chlorophyll *a* fluorescence was reported as the Fv/Fm ratio, which shows the maximal quantum yield of PSII photochemistry, where Fm = the maximal fluorescence and Fv = the variable fluorescence.

#### *2.4. Estimation of Proline Content*

The protocol of Bates et al. [48] was used for determining the proline content, using L-proline as the standard. In total, 1 g of fresh leaf samples was homogenized in 5 mL of 3% aqueous sulfosalicylic acid and centrifuged at 14,000× *g* for 15 min (Neya 10R, REMI, Mumbai, India). The supernatant (2 mL) was blended with an equal volume of acetic acid and acid ninhydrin, vortexed, and incubated for 1 h at 100 ◦C. An ice bath was used to stop the reaction, and the solution was extracted with 4 mL of toluene. The sample was read spectrophotometrically at 525 nm using toluene as the blank (7315 Spectrophotometer, Jenway, Staffordshire, UK).

#### *2.5. Extraction and Assay of Antioxidant Enzymes*

In total, 0.5 g fresh leaf sample was extracted with 4 mL of buffer (50 mM potassium phosphate, 1 mM EDTA, 1% PVP, 1 mM DTT and 1 mM PMSF, pH 7.8). The samples were centrifuged at 15,000× *g* for 30 min at 4 ◦C [49]. The supernatant was picked up and stored at −80 ◦C for the determination of enzyme activity. The catalase activity (CAT) was measured via the protocol of Aebi [50]. Then, 20 μL of supernatant was blended with 830 μL of potassium phosphate buffer (50 mM, pH 7). The reaction was initiated by the addition of 150 μL of H2O2, and we registered the decrease in absorbance (240 nm) for 2 min. The GPX activity was determined using the protocol described by Ruley et al. [34]. A reaction mixture consisting of suitable quantities of enzymatic extracts was employed, with equal amounts of 17 mM H2O2 and 2% guaiacol. The reaction was registered for 3 min at 510 nm. The Giannopolitis and Ries [51] protocol was used for determining the SOD activity (560 nm).

#### *2.6. Climate Conditions*

The climate conditions (air temperatures (◦C) and relative humidity (RH %)) in both cycle periods were registered using a CR 1000 data logger (Campbell Scientific Ltd., Loughborough, UK). The mean air temperatures during I CP and II CP were 13.4 ◦C and 21.4 ◦C, respectively, and the mean relative humidity values were 86% and 76.0%, respectively (Figure S1).

#### *2.7. Statistical Analysis*

Both experimental trials were conducted via a randomized complete design; three replicates in each cycle period (CP) and for each treatment (T) were used. Statistical analysis of the data was performed using CoStat release 6.311 (CoHort Software, Monterey, CA, USA). The data were subjected to two- and one-way analysis of variance (ANOVA), and the means were compared via a Tukey's test at *p* < 0.05. The data presented in the figures are the means ± standard error (SE).

#### **3. Results**

Table 1 shows the main effects and the interaction effects (over cycle periods and treatments) of morphometric parameters and mineral contents. The two-way ANOVA showed that the parameters were influenced by cycle and treatments (Table 1). The total and shoot dry biomass were statistically different in different cycle periods. Significant differences were found in the root/shoot ratio depending on the two factors and their interactions. Leaf number and total leaf area were only statistically significant for the cycle periods. There were no significant differences in chlorophyll content for any factors. The mineral contents in the leaves and in the roots were significantly different between cycle periods and treatments, and their interactions had an effect too. Leaf damage was only significantly different between treatments. To better understand the effects of the cycle periods on the treatments, the two cycle periods were separately analyzed.

**Table 1.** Summary of the main effects and interaction effects of cycle periods (I CP and II CP) and treatments (control, salt spray, surfactants, and salt plus surfactants) on total and epigeous dry biomass, root/shoot ratio, leaf number, total leaf area, chlorophyll content, Na+ and Cl− in leaves and roots, and leaf damage of *Callistemon* plants with the corresponding significance of the F-values.

Significance of differences in parameters: ns = not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 with the corresponding significance of the F-values.

Salt spray, alone or in combination with the surfactant, significantly influenced the biomass accumulation in both growing periods (Table 2). The total dry biomass was reduced in I CP under all treatments by 30% in relation to the control plants (Figure 1a). During II CP, the total dry biomass was only reduced in the treatment with salt by 33% (Table 2). Shoot dry biomass showed a similar trend in both cycle periods (Table 2).

All treatments slightly modified root dry biomass (data not shown), and in I CP, enhanced the root/shoot ratio (Table 2).

The effects of treatments on leaf numbers and total leaf area were observed in both cycle periods (Table 2). Leaf number and total leaf area were significantly reduced during I CP in all treatments, with a reduction of 64% in relation to the control plants (Table 2). A similar trend was registered during II CP, but with only a minor reduction for both parameters (by 20% and 14% for leaf number and total leaf area, respectively).

The chlorophyll contents in both cycle periods did not change according to the treatments (Table 2).

At the end of I CP, a seven-fold higher leaf Na+ content was registered in plants treated with salt spray and salt spray plus surfactants in relation to the control plants and those treated with surfactants only. In II CP, this increase was amplified; 8- and 11-fold increases were noted in plants treated with salt spray and salt spray plus surfactants, respectively (Table 2).

A similar trend was registered for the leaf Cl− content; at the end of the I CP, a five-fold higher Cl− content was noted in the plants treated with salt spray and with salt spray plus surfactants in relation to the control plants and those treated with surfactants only. In CP II, we noticed a six-fold increase in plants treated with salt spray and salt spray plus surfactants (Table 2).

No significant differences were observed in the root mineral content during I CP. In II CP, on the other hand, an increase in the Na+ content by 33% was observed in plants treated with salt spray and by 44% in those treated with salt plus surfactants. The Cl− content increased by 50% in plants treated with salt spray and salt spray plus surfactants (Table 2).

**Figure 1.** Net photosynthesis (AN) (**a**,**b**) and leaf stomatal conductance (gs) (**c**,**d**) in Callistemon plants during I CP and II CP. Plants were subjected twice a week to nebulization treatments for 8 weeks, depending on their treatments: Control (deionized water), salt spray (solution simulating the composition of seawater), surfactants (sodium dodecylbenzene-sulfonate), and a solution with seawater and anionic surfactant. Mean values ± SE (*n* = 4).

The leaf damage percentages were significantly different among treatments. During I CP, the average leaf damage percentage was 18% in plants treated with salt spray and salt spray plus surfactants. During II CP, the maximum value (~27%) was observed in salt spray plus surfactants plants; no leaf damage was observed in plants treated with only surfactants in either cycle period (Table 2).

Values are the means ± SE of three replicate samples. n.s. = not significant; \*, \*\*, \*\*\* significant at *p* < 0.05, 0.01 and *p* < 0.001, respectively. The values in the same column followed by the same letter are not significantly different at *p* ≤ 0.05 (Tukey's test). The gas exchange in both cycle periods was significantly modified as a result of the treatments (Figure 1). From the first measurement (2 weeks), *Callistemon* plants in I CP treated with salt spray alone or in combination with surfactants showed reductions of 33% and 50% in AN, compared with the control plants; at the end of I CP, a reduction of 23% in this activity in salt spray and salt spray and surfactant plants was observed (Figure 1a). The stomatal conductance values during I CP showed no significant differences (Figure 1c).

During II CP, the reduction in photosynthesis activity was significant immediately after the second week, and this trend was maintained until the end of the experimental period. Under control conditions, the net photosynthesis was about 5 μmol CO2 m−2 s−1. Salt spray alone or in combination caused a progressive decrease in net photosynthesis assimilation; at the end of the cycle period, a reduction of ~60% was reached (Figure 1b). A similar response was registered in the stomatal conductance. During the warmer season, stomatal conductance showed higher values throughout the growth cycle, but a more

intense reduction was seen at the end of the trial in the salt spray plus surfactants group (~60%) (Figure 1d), compared with the control plants.

The different values of chlorophyll *a* fluorescence (Fv/Fm ratio) after exposure to different treatments in different growth periods are presented in Figure 2a,b. The plants treated with salt spray and salt spray plus surfactant showed reductions in both cycle periods, compared with the control plants; the Fv/Fm achieved respective values of 0.67 and 0.71 in I CP, and 0.67 and 0.65 during II CP.

**Figure 2.** Maximum quantum efficiency of the PSII (Fv/Fm) of *Callistemon* plants during I CP (**a**) and II CP (**b**) treated with distilled water (control), salt spray, surfactants, and salt plus surfactants. Values are means ± SE (*n* = 3).

In the final part of the experiment, in both cycle periods, the leaf proline content increased in the salt spray and salt plus surfactants treatment groups, compared with the control and surfactants plants, in both cycle periods, by 65% and 37% in I CP, and 75% and 63% in II CP (Figure 3).

**Figure 3.** Proline contents in leaves of *Callistemon* during I CP ( ) and II CP ( ) treated with distilled water (control, and ), salt spray ( and ), surfactants ( and ), and salt plus surfactants ( and ). Values are means ± SE (*n* = 3). Values with the same letter are not significantly different as determined by Tukey's test (*p* < 0.05).

The enzyme activities, SOD, CAT, and GPX, exhibited significant differences in the cycle periods, depending on treatments (Figure 4).

**Figure 4.** Effects of different treatments on catalase (CAT (**a**)), peroxidase (GPX (**b**)), and superoxide dismutase (SOD (**c**)) activity in *Callistemon* plants during I CP (( ) and II CP ( ) treated with distilled water (control, and ), salt spray ( and ), surfactants ( and ), and salt plus surfactants (( and ). Values are means ± SE (*n* = 3). Values with the same letter are not significantly different by Tukey's test (*p* < 0.05).

The CAT activity at the end of I CP showed no significant differences. In II CP, however, the salt spray and the salt spray plus surfactants treatment groups showed significant increases in this enzyme by 47% compared with the control and surfactants treatments (Figure 4a).

The GPX activity during I CP was lower in the surfactants compared with the other treatments (Figure 4b). In the final part of II CP, however, the salt treatments increased the activity of this enzyme by 42% and 12% in the salt and salt plus surfactants treatments, respectively (Figure 4b).

In I CP, the SOD activity was much lower and showed its highest values in the salt treatments, with an increase of 20%. In the second cycle period, the observed values were much higher and were again even higher in the salt treatment groups, with increases of 21% and of 42% in the salt spray and salt plus surfactants groups, respectively (Figure 4c).

To visualize the associations among the different treatments in terms of the plants' morphological, physiological, and biochemical parameters in the two cycle periods, a principal component analysis was performed for all analyzed parameters (PCA; Figure 5a,b; Tables S1 and S2). The first two principal components (PC1 and PC2) accounted for 59% and 28% and 73% and 19% of the total variance during I CP and II CP, respectively, and these components were associated with eigenvalues higher than one.

**Figure 5.** Principal component loading plot and scores of the PCA on the total and shoot dry biomass, R/S, leaf number, total leaf area, chlorophyll content (Chl), Na+ leaf and root content, Cl− leaf and root content, leaf damage, gas exchange (AN and gs), Fv/Fm, proline (Pro), and enzyme activity (GPX, CAT, and SOD), for *Callistemon* plants undergoing different salt treatments (control, salt spray, surfactants, and salt plus surfactants) according to the first two principal components in I CP (**a**) and II CP (**b**).

During I CP, PCA revealed that the morphological and physiological parameters were directly affected by the control and surfactants treatments, whereas the mineral contents and the biochemical parameters were directly correlated with the salt and salt plus surfactants treatments (Figure 5a).

A similar trend was found at the end of II CP (Figure 5b).

#### **4. Discussion**

The negative influence of salinity on ornamental plants has been studied. Nevertheless, only a limited number of papers have provided information on the effects of salt spray alone or in combination with surfactants. The identification of plants tolerant to salt spray is particularly important in the selection of shrubs and trees to be used in green areas [9].

A large number of studies on salt stress have used NaCl as the salinizing agent [52–54]. However, the compositions of salts in seawater are rather different. Saline solutions containing a single salt may give rise to misleading and erroneous interpretations of plants' responses to salinity [55,56]. In our studies, we have added to deionized water a solution reproducing the composition of seawater [46] so as to simulate the effects of salt spray along coastal areas.

Salt spray can hinder plant growth because it causes drought stress, disrupts membranes and enzyme systems, reduces nutrient uptake, causes necrosis or leaf loss, and can lead to plant mortality [15].

In both cycle periods, the total and shoot dry biomass were reduced in the presence of salt spray, alone or in combination with surfactants. Fragmented cuticles, disaggregated chloroplasts, and nuclei disrupted stomata, collapsed cell walls, coarsely granulated cytoplasm, and disorganized phloem leading to reduced biomass were observed after the application of spray NaCl [57].

Salt treatments reduced the epigeous dry biomass, reduced the leaf number and total leaf area, and did so more extensively during II CP. This behavior confirms that the plants reduce their leaf area to overcome stress [58]. Plants implement different mechanisms to overcome abiotic stress (drought, saline stress), including a reduction in the total leaf area, which leads to a reduction in water losses through stomata closure, which is the principal defense strategy of species subject to osmotic stress [12].

Reductions in leaf area can therefore be a strategy of adaptation. The reduction in this parameter is generally related to a decrease in the total leaf number and size. In the presence of abiotic stress (drought and salt stress), plants reduce their leaf size so as to reduce the surface available for both the depositing of salt and the loss of water through transpiration [59]. Reduction in leaf number was observed in plants of *Scaevola sericea* [60] and *Crambe maritime* [61] sprayed with seawater. This agrees with the results of our study. The plants grown during II CP and treated with salt spray showed a greater reduction in leaf number. This result agrees with the experiments of Bussotti et al. [41], who found that the damage of surfactants was greater in the warmer season.

Aesthetic quality is the most important trait in ornamental plant species [62]. Visual quality is a practical tool used to evaluate the salt response of ornamental species [63]. Common responses in the presence of salt stress include leaf necrosis and reductions in chlorophyll [64]; greater sensitivity has been observed in older leaves [65]. In our study, leaf necrosis as a symptom of leaf damage was registered, and this ranged from minimal (surfactant) to moderate (salt spray and salt spray plus surfactants). Leaf damage could lead to a reduction in the photosynthetic rate, which might manifest in diminished growth [66].

A potential biomarker of salinity tolerance is indicated by the absorption and accumulation of ions in the leaves [2]. The Na+ contents significantly increased during salt treatments in both cycle periods, although this was greater during the summer cycle, and the presence of surfactants intensified this effect further still. In this case, the presence of surfactants increased the phytotoxic effects, as reported by Sànchez-Blanco et al. [67]. High-rated absorptions of Na+ and Cl− in the shoots reduce growth rate due to ion toxicity. Excessive Na and Cl accumulation leads to molecular damage-causing growth arrest and plant death because these ions induce cytoplasmic toxicity [68].

Different studies suggest ornamental plants exposed to salt treatments show a reduction in net photosynthesis and stomatal conductance. In our work, salt treatments, alone or in combination with surfactants, caused a reduction in net photosynthesis as a consequence of the reduced CO2 assimilation following stomatal closure, which particularly occurred during II CP.

The reduction in Fv/Fm is a clear signal that the PSII was affected by the presence of salt and that photoinhibition occurred. Generally, plants under normal conditions presented Fv/Fm ratios close to 0.8 [7], but in our case, the reduction in Fv/Fm was correlated with salt spray. The plants showed a reduction in this index in the presence of salt, and this reduction was greater when the salt was combined with a surfactant. For different plant species, regardless of whether the PSII damages occur in the leaves, this index is considered one of the most effective tools for the measurement of plant stress response, particularly before any morphological changes manifest [69]. The reduction in photosynthetic capacity is correlated with reductions in chlorophyll fluorescence in ornamental species; these cause physiological modifications that will lead to losses in sales for the growers [70].

One of the mechanisms adopted to defend against salt stress is proline accumulation, which can be used as a selection criterion for salt tolerance because an increase in this amino acid may be positively correlated with the level of salt tolerance [71]. In fact, modifications in soluble protein and proline content could regulate cellular redox potential [72] and help scavenge free radicals [73].

Increased ROS production, which damages biomolecules (e.g., lipids, proteins, and nucleic acids) and harms redox homeostasis, was observed in the presence of salt stress [74]. The increase in ROS activity causes an increase in oxidative stress due to ionic toxicity and osmotic imbalance and has negative effects on photosynthetic pigments [75]. In the presence of saline stress, plants produce reactive oxygen species (ROS), such as hydroxyl radical (OH•), singlet oxygen (1O2), superoxide radical (O2 •−), and hydrogen peroxide (H2O2), which have a toxic effect on plants [76]. Our results show that the enzyme activity was similar between the control and surfactant treatments but was significantly increased under the salt spray treatment. These results showed that *Callistemon* plants have significant salt tolerance, and as reported by Sklodowska et al. [77], a greater increase in SOD activity could help preserve membrane integrity and osmoregulation by helping regulate the level of superoxide radical in the presence of high saline levels.

To better understand the tolerance mechanisms related to salt stress and surfactants, it will be relevant to investigate and understand the morphological, physiological, and biochemical parameters of plants grown under stressed and not-stressed conditions (Supplementary Tables S1 and S2). To evaluate stress tolerance and the relationships among the different parameters analyzed in our study, an effective approach is the analysis of principal components (PCA) [12]. Our PCA results demonstrate that the variations in stress tolerance between *Callistemon* during I CP and II CP are linked to variations in biochemical and mineral contents.

#### **5. Conclusions**

Our trial has demonstrated the combined effects of salt spray and surfactant on the ornamental shrub species largely used in Mediterranean green areas near the sea. Salt spray, alone or in combination with surfactant action, influences plants' growth and aesthetic features, especially during warmer seasons when transpiration is more intense and causes stomatal opening. To overcome stressful conditions, plants undergo several changes, such as the activation of enzymes and the accumulation of proline and minerals (Na+ and Cl−). Interaction with surfactants amplifies these adverse effects.

The results of this trial help us to better understand the responses of plant species used in green areas near the coastline and will thus guide the choice of plant species.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8030261/s1, Figure S1: Air temperature (◦C) and relative humidity (%) during the two cycle periods, Table S1: Pearson's correlation coefficients among morphometric, physiological, and biochemical parameters from *Callistemon* potted plants exposed to different treatments during I CP, Table S2: Pearson's correlation coefficients among morphometric, physiological, and biochemical parameters from *Callistemon* potted plants exposed to different treatments during II CP.

**Author Contributions:** Conceptualization, D.R. and S.T.; methodology, D.R. and S.T.; software, S.T.; validation, D.R.; formal analysis, S.T. and G.L.F.; investigation, S.T. and G.L.F.; resources, D.R.; data curation, S.T.; writing—original draft preparation, D.R. and S.T.; writing—review and editing, D.R. and S.T.; supervision, D.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data for this study has been included in the tables, figures, and Supplemental Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

#### MDPI

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**3. Background**

2 Will-be-set-by-IN-TECH

(2007) and Francavilla et al. (2011), perfectly addresses our requirements (see Section 3.4 for details). In this chapter, a Grid Computing infrastructure is presented. This architecture allows parallel block execution by distributing tasks to nodes that belong to the Grid. The set of nodes is composed by physical machines and virtualized ones. This feature enables great flexibility and increase available computational power. Furthermore, the presence of virtual nodes allows a full and efficient Grid usage, indeed the presented architecture can be

The chapter is organized as follow, Section 2 briefly explains author's contribution. Section 3 describes technologies used and summarized Domain Decomposition principles. The architecture is shown in Section 4, while the advantages derived by its adoption are illustrated

Due to the decomposition of the original problems into a larger number of subproblems, the scheme is well suited to a parallelization approach, since the subproblems (which will be referred to as blocks in the following) are disjoint, and the elaboration of the blocks is intrinsically a parallelizable operation. Since the generation of the entire domain basis functions on each block is independent from the other blocks, a high scalability is expected. As an example, Figure 1 shows a fighter subdivided into 4 different blocks, identified by different colors. The four blocks can be processed in parallel by four different processes, in order to generate the basis functions describing each block. Parallelization can be achieved using two different techniques: parallel programming or Grid Computing. Parallel programming is the technique by which have been obtained the best results in this field (500 billion of unknowns) Mouriño et al. (2009). There are important barriers to the broader adoption of these methodologies though: first, the algorithms must be modified using parallel programming API like MPI (2011) and OpenMP (2011), in order to run jobs on selected cores. The second aspect to consider is the hardware: to obtain results of interest supercomputers with thousands of cores and thousands of GB of RAM are required. Typically these machines are made by institutions to meet specific experiments and do not always allow public access. Grid Computing is a rather more easily applicable model for those who do not fulfill the requirements listed above Foster & Kesselman (2003). For its adoption the only requirement is to have at disposal computers to use within the grid. The machines may be heterogeneous, both in terms of the types (workstations, servers) and hardware resources of each machine (RAM, CPU, HD); besides they can also be recovery machines. With the Virtualization technology the number of processes running on each machine (for multicore ones) can be optimized by instantiating multiple virtual nodes on the same physical machine. The real advantage of this model, however, is that researchers do not have to worry about rewriting code to make their application parallelizable. One of the drawbacks in the adoption of this model is the addition of overhead introduced by the grid (e.g., inputs time transfer) and by the hypervisor that manages the virtual machines. It has been shown, however, that the loss of performance due to the hypervisor is not particularly high, usually not exceeding 5% Chierici & Verald (2010). For the above reasons, in our case it was decided to grid infrastructure, composed of heterogeneous recovery machines, both physical and virtual, on which to run scientific applications without the need to modify the source codes of the used algorithms.

in Section 5 and Section 6 draws conclusions and gives hints for future works.

The ultimate goal is the reduction of execution times of CEM applications.

used by different users that run different applications.

**2. Motivation**

The rapid technology growth in the recent years has helped the development and rapid expansion of Grid Computing. "The roots of Grid Computing can be traced back from the late 80s when the research about scheduling algorithms for intensive applications in distributed environments accelerated considerably" Kourpas (2006). In the late 1990s began to emerge, a more generalized framework for accessing high performance computing systems, and at the turn of the millennium, the pace of change was accelerated by the recognition of potential synergies between the grid and the emerging Service Oriented Architectures (SOA) through the creation of the Open Grid Services Architecture (OGSA) Kourpas (2006). "The Open Grid Services Architecture is a set of standards defining the way in which information is shared among several components of large, heterogeneous grid systems. The OGSA is, in effect, an extension and refinement of the Service Oriented Architecture" OGSA (2007). The Open Grid Services Architecture is a standard created by the Open Grid Forum (OGF) OGF (2011). OGF was founded in 2006 from the Global Grid Forum (GGF) and the Enterprise Grid Alliance (EGA). GGF had a rich background and established international presence within the academic and research communities while EGA was focused on developing and promoting enterprise grid solutions. The OGF community now counts thousands of members working in research and industry, representing more than 400 organizations in 50 countries.


**1. Introduction**

A classical method to characterize the spectral density of phase noise of microwave oscilla‐ tors is to compare the device under test (DUT) to another one, we call it the reference, with the same frequency if noise is expected to be better for the reference. In most of case it is possible to then characterize oscillators or synthesizers. But it is only possible if we can as‐ sign the same frequency to the DUT and the reference signal. However for some applica‐ tions, we see that the DUT delivers a frequency hard to predict during the fabrication. We here focus on characterizing a special class of oscillators, called optoelectronic oscillators (OEO) [1]. An OEO is generally an oscillator based on an optical delay line and delivering a microwave signal [2]. Purity of microwave signal is achieved thanks to a delay line inserted into the loop. For example, a 4 km delay corresponds to a 20 μs-long storage of the optical energy in the line. The continuous optical energy coming from a laser is converted to the mi‐ crowave signal. This kind of oscillators were investigated [3]. By the way, such OEO based on delay line are still sensitive to the temperature due to the use of optical fiber. Recently, progress were made to set compact OEO thanks to optical mini-resonators or spheres [4 - 7]. Replacing the optical delay line with an ultra-high Q whispering gallery-mode optical reso‐ nator allows for a more compact setup and an easier temperature stabilization. In order to introduce into the loop the fabricated resonator in MgF2 [8], CaF2 or fused silica, it has to be coupled to the optical light coming from a fiber. Best way to couple is certainly to use a cut optical fiber through a prism. But a good reproducible way in a laboratory is to use a ta‐ pered fibber glued on a holder. Then appears a problem for determining the phase noise of such compact OEO, because the frequency is rarely predictable. It is impossible to choose the frequency of the modulation (for instance exactly 10 GHz) and that's why it is necessary to develop new instruments and systems to determine phase noise for any delivered signal

© 2013 Salzenstein; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Salzenstein; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in X-band (8.2-12.4 GHz) for instance. Here we come to our goal. The aim of this chapter is to provide a tool for a better knowledge of the phase noise characterization of OEO's using an optoelectronic phase noise system. We logically start by giving the main principle of how work such a system. We see that the main idea is to use delay lines to perform phase noise measurements. We also consderably increase the performances of such a system by cross correlation measurement, thanks to the two quasi-identical arms developed instrument. Then we present a realized system and the evaluation of its uncertainty based on interna‐ tional standard for determination of uncertainties when characterizing an OEO in X-band.

#### **2. Principle of the phase noise measurement system**

A quasi-perfect RF-microwave sinusoidal signal can be written as :

$$\mathbf{U}\cdot\boldsymbol{\nu}(t) = V\_0 \mathbf{I} + \alpha(t) \mathbf{J} \cos\{2\pi\nu\_0 t + \boldsymbol{\varphi}(t)\}\tag{1}$$

Reference is no more required for homodyne measurement with a delay line discriminator. At microwave frequencies, electrical delay is not suitable because of its high losses. Howev‐ er photonic delay line offers high delay and low attenuation equal to 0.2 dB/km at the wave‐ length λ=1.55 μm. Optoelectronic phase noise measurement system is schematically

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339

It consists on two equal and fully independent channels. The phase noise of the oscillator is determined by comparing phase of the transmitted signal to a delayed replica through opti‐ cal delay using a mixer. It converts the phase fluctuations into voltage fluctuations. An elec‐ tro-optic modulator allows modulation of the optical carrier at microwave frequency. The length of the short branch where microwave signal is propagating is negligible compared to the optical delay line. Mixers are used as phase detectors with both saturate inputs in order to reduce the amplitude noise contribution. The low pass filters are used to eliminate high frequency contribution of the mixer output signal. DC amplifiers are low flicker noise.

The oscillator frequency fluctuation is converted to phase frequency fluctuation through the delay line. If the mixer voltage gain coefficient is Kφ (volts/radian), then mixer output rms

Where │Hφ(jf)│² = 4.sin²(πfτ) is the transfer function of optical delay line, and f is the offset frequency from the microwave carrier. Equation (3) shows that the sensitivity of the bench depends directly on K²φ and │Hφ(jf)│. The first is related to the mixer and the second es‐ sentially depends on the delay τ. In practice, we need an FFT analyzer to measure the spec‐ tral density of noise amplitude V²out(f)/B, where B is the bandwidth used to calculate Vout(f)/B. The phase noise of the DUT is finally defined by Eq. (4) and taking into account the

*V* ²*out* ( *f* ) = *K*²*φ* │*Hφ*( *jf* )│²*Sφ*( *f* ) (3)

represented on Figure 1.

**Figure 1.** Phase noise bench.

voltage can be expressed as :

gain of DC amplifier GDC as :

where V0 is the amplitude, ν0 is the frequency, α(t) is the fractional amplitude fluctuation, and φ(t) is the phase fluctuation. Equation (1) defines α(t) and φ(t) in low noise conditions: | α(t)| <<1 and |φ(t)| <<1. Short-term instabilities of signal are usually characterized in terms of the single sideband noise spectral density PSD S(f). Phase noise £(f) is typically expressed in units of dBc/Hz, representing the noise power relative to the carrier contained in a 1 Hz bandwidth centered at a certain offsets from the carrier. So, S is typically expressed in units of decibels below the carrier per hertz (dBc/Hz) and is defined as the ratio between the oneside-band noise power in 1 Hz bandwidth and the carrier power:

$$
\varepsilon(f) = \mathbb{W} \cdot \operatorname{Sq}(f) \tag{2}
$$

This definition given in equation (2) includes the effect of both amplitude and phase fluctua‐ tions. Phase noise is the frequency domain representation of rapid, short-term, random fluc‐ tuations in the phase of a waveform, caused by time domain instabilities. However we must know the amplitude and phase noise separately because they act differently in the circuit. For example, the effect of amplitude noise can be reduced by amplitude limiting mechanism and mainly suppressed by using a saturated amplifier. Phase noise of microwave oscillators can usually be characterized by heterodyne measurement. Whereas, for such a system, we need a reference oscillator operating exactly at the frequency of the DUT with lower phase noise. Phase noise can be measured using a spectrum analyzer if the phase noise of the de‐ vice under test (DUT) is large with respect to the spectrum analyzer's local oscillator. Care should be taken that observed values are due to the measured signal and not the Shape Fac‐ tor of the spectrum analyzer's filters. Spectrum analyzer based measurement can show the phase-noise power over many decades of frequency. The slope with offset frequency in vari‐ ous offset frequency regions can provide clues as to the source of the noise, e.g. low frequen‐ cy flicker noise decreasing at 30 dB per decade.

Reference is no more required for homodyne measurement with a delay line discriminator. At microwave frequencies, electrical delay is not suitable because of its high losses. Howev‐ er photonic delay line offers high delay and low attenuation equal to 0.2 dB/km at the wave‐ length λ=1.55 μm. Optoelectronic phase noise measurement system is schematically represented on Figure 1.

**Figure 1.** Phase noise bench.

in X-band (8.2-12.4 GHz) for instance. Here we come to our goal. The aim of this chapter is to provide a tool for a better knowledge of the phase noise characterization of OEO's using an optoelectronic phase noise system. We logically start by giving the main principle of how work such a system. We see that the main idea is to use delay lines to perform phase noise measurements. We also consderably increase the performances of such a system by cross correlation measurement, thanks to the two quasi-identical arms developed instrument. Then we present a realized system and the evaluation of its uncertainty based on interna‐ tional standard for determination of uncertainties when characterizing an OEO in X-band.

where V0 is the amplitude, ν0 is the frequency, α(t) is the fractional amplitude fluctuation, and φ(t) is the phase fluctuation. Equation (1) defines α(t) and φ(t) in low noise conditions: | α(t)| <<1 and |φ(t)| <<1. Short-term instabilities of signal are usually characterized in terms of the single sideband noise spectral density PSD S(f). Phase noise £(f) is typically expressed in units of dBc/Hz, representing the noise power relative to the carrier contained in a 1 Hz bandwidth centered at a certain offsets from the carrier. So, S is typically expressed in units of decibels below the carrier per hertz (dBc/Hz) and is defined as the ratio between the one-

This definition given in equation (2) includes the effect of both amplitude and phase fluctua‐ tions. Phase noise is the frequency domain representation of rapid, short-term, random fluc‐ tuations in the phase of a waveform, caused by time domain instabilities. However we must know the amplitude and phase noise separately because they act differently in the circuit. For example, the effect of amplitude noise can be reduced by amplitude limiting mechanism and mainly suppressed by using a saturated amplifier. Phase noise of microwave oscillators can usually be characterized by heterodyne measurement. Whereas, for such a system, we need a reference oscillator operating exactly at the frequency of the DUT with lower phase noise. Phase noise can be measured using a spectrum analyzer if the phase noise of the de‐ vice under test (DUT) is large with respect to the spectrum analyzer's local oscillator. Care should be taken that observed values are due to the measured signal and not the Shape Fac‐ tor of the spectrum analyzer's filters. Spectrum analyzer based measurement can show the phase-noise power over many decades of frequency. The slope with offset frequency in vari‐ ous offset frequency regions can provide clues as to the source of the noise, e.g. low frequen‐

*υ*(*t*)=*V*0 1 + *α*(*t*) *cos*(2*πν*0*t* + *φ*(*t*)) (1)

£( *f* ) = ½⋅ *Sφ*( *f* ) (2)

**2. Principle of the phase noise measurement system**

338 Optoelectronics - Advanced Materials and Devices

A quasi-perfect RF-microwave sinusoidal signal can be written as :

side-band noise power in 1 Hz bandwidth and the carrier power:

cy flicker noise decreasing at 30 dB per decade.

It consists on two equal and fully independent channels. The phase noise of the oscillator is determined by comparing phase of the transmitted signal to a delayed replica through opti‐ cal delay using a mixer. It converts the phase fluctuations into voltage fluctuations. An elec‐ tro-optic modulator allows modulation of the optical carrier at microwave frequency. The length of the short branch where microwave signal is propagating is negligible compared to the optical delay line. Mixers are used as phase detectors with both saturate inputs in order to reduce the amplitude noise contribution. The low pass filters are used to eliminate high frequency contribution of the mixer output signal. DC amplifiers are low flicker noise.

The oscillator frequency fluctuation is converted to phase frequency fluctuation through the delay line. If the mixer voltage gain coefficient is Kφ (volts/radian), then mixer output rms voltage can be expressed as :

$$V^2\_{\
uat} \begin{Bmatrix} f \end{Bmatrix} = K^2\_{\varphi} \begin{Bmatrix} H\_{\varphi} \begin{Bmatrix} \dot{f} \end{Bmatrix} \end{Bmatrix} \begin{Bmatrix} \dot{f} \end{Bmatrix} \begin{Bmatrix} f \end{Bmatrix} \tag{3}$$

Where │Hφ(jf)│² = 4.sin²(πfτ) is the transfer function of optical delay line, and f is the offset frequency from the microwave carrier. Equation (3) shows that the sensitivity of the bench depends directly on K²φ and │Hφ(jf)│. The first is related to the mixer and the second es‐ sentially depends on the delay τ. In practice, we need an FFT analyzer to measure the spec‐ tral density of noise amplitude V²out(f)/B, where B is the bandwidth used to calculate Vout(f)/B. The phase noise of the DUT is finally defined by Eq. (4) and taking into account the gain of DC amplifier GDC as :

$$\mathbb{E}\{f\} = \left[V^2\mathbb{I}\_{\text{out}}\{f\}\right] \cdot \left|\cdot\right| \cdot \left[\mathbb{Z}K^2\mathbb{I}\_{\text{\textquotedblleft}\emptyset}\cdot \left|\,H\_{\text{\textquotedblleft}\{\mathcal{J}\}}\right|\right|\,^2G^2\_{\text{DC}}\cdot\mathbb{B}\right] \tag{4}$$

**Figure 3.** Picture of the double channels *Hewlett-Packard* HP3561A FFT analyzer.

[11] with the phase noise we measure using our system.

The measured phase noise includes the DUT noise and the instrument background. The cross correlation method allows to decrease the cross spectrum terms of uncommon phase noise as √(1/m), where m is the average number. Thereby uncorrelated noise is removed and sensitivity of measure is improved. To validate the measure of our phase noise bench, we need to compare data sheet of the commercial frequency synthesizer Anritsu/Wiltron 69000B

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**Figure 4.** Phase noise (dBc/Hz) of the synthesizer measured at 10 GHz with Kφ=425 mV/rad and GDC= 40dB versus

**4. Validation of the performances**

Fourier Frequency between 10 Hz and 100 kHz.

Such instruments has been recently introduced [3,9,10]. In section 3, we present concretely a realized optoelectronic phase noise measurement system.