Pre-visual detection of iron and phosphorus deficiency by transformed reflectance spectra

Abstract

Reflectance spectroscopy and strategies for spectral analysis over the visible range from 380 to 780 nm were used to provide diagnostic information on iron (Fe) and phosphorus (P) status of Brassica chinensis L. var parachinensis (Bailey) grown under hydroponics conditions. Leaf reflectance (R) spectra were collected and normalized inner reflectance (NR^I) spectra were calculated. The regression coefficients (B-matrix) and variable importance for projection (VIP) in partial least squares regression were used to determine important wavelengths that correlate with total chlorophyll (Chl) content. No single wavelength that showed good correlation with Chl content was found. Therefore, NR^I was transformed into CIELAB color values, which simplified the whole visible spectrum into three values. Our results showed that upon Fe deprivation, plants entered into a deficiency state very rapidly, highlighting the importance of early diagnosis. The direct effect of Fe on leaf Chl content allowed CIELAB color values to be used for pre-visual detection of Fe deficiency 2 days before the appearance of visually distinguishable morphological changes. On the other hand, P-deprived plants showed a marked decline in cellular P levels but remained above critical threshold concentrations after 7 days. The Chl content was not affected by the leaf P content and CIELAB color values showed no difference with control plants.

Introduction

Sub-optimal management of crop nutrient poses a major yield constraint in commercial crop production for food, medicine or valuable plant extracts. Optimal mineral nutrient management is also of key importance for the production of quality food to meet human nutritional requirements [1]. Conventional approaches to ascertaining plant nutrient status rely primarily on determining total elemental content in plant tissues and quick field tests on plant sap and fluids [2]. These methods are time consuming, tedious, expensive and involve destructive sampling of plant parts. A further disadvantage of conventional methods is that the interpretations of total elemental content in tissues do not generally include consideration of the distribution and form of the element within cells. For example, iron (Fe) can exist in trivalent or divalent forms in plant tissues or complexed within Fe-containing proteins and enzymes, thus influencing its bioavailability for metabolic reactions and chlorophyll biosynthesis. In lettuce exhibiting distinct iron-deficient symptoms, 93–604 μg g⁻¹ Fe was found in the tissues compared with 130–1468 μg g⁻¹ Fe presented in healthy plants [3]. This phenomena has also been observed in Spathiphyllum where Fe-deficient leaves manifesting interveinal chlorosis contained Fe at levels similar to that of Fe-sufficient leaves exhibiting no morphological symptoms [4], [5]. Hence, simple analysis of total elemental content may be of limited value for evaluation of nutrient adequacy within plant tissues in some cases.

Fe mineral nutrition presents difficulties in crop management due to the low solubility of both ferrous (Fe²⁺) and ferric (Fe³⁺) iron compounds, particularly in calcareous soils [6]. Amendment of soil pH may also cause available Fe to precipitate due to overliming, thus leading to Fe deficiency or lime-induced chlorosis. Plant response to Fe deficiency is manifested in a plethora of morphological and biochemical changes [4], [7], [8], [9]. Typical consequences of iron-deficient (–Fe) states include interveinal chlorosis of apical leaves and the structural disorganization of chloroplasts and mitochondria [10], [11]. Besides yellowing, plant growth can also be hampered [8]. In severe cases, Fe deficiency can result in extensive chlorosis, necrosis, arrested growth or even death.

Phosphorus (P) is a key macronutrient absorbed by plants mostly in the monovalent orthophosphate form ${\mathrm{H}}_2{\mathrm{PO}}_4^{-}$. Under cytoplasmic pH conditions, inorganic phosphate (P_i) remain largely as the primary or secondary orthophosphate anions, ${\mathrm{H}}_2{\mathrm{PO}}_4^{-}\mathrm{and}{\mathrm{HPO}}_4^{2-}$. Intracellular P can be complexed into organic forms as a structural component of key macromolecules including nucleic acids, phospholipids, certain amino acids and coenzymes. The linkage of three P_i molecules via a phosphoester and two phosphoanhydride bonds to form high energy ATP molecules contributes to the significant role of P in energy transfer and in providing the necessary metabolic energy in photosynthesis and respiration [12], [13], [14], [15]. The symptoms of P deficiency can be explained on the basis of its multifarious functional roles in plants. Since P is an essential component of genetic material, cell division and expansion are adversely affected by P deprivation [15] and P-deficient (–P) plants typically display weak or stunted growth. Lack of P also hinders metabolic processes leading to sugar buildup, which is associated with anthocyanin formation and the development of a purplish tinge in leaves and stems. P is not a component of chlorophyll but P deficiency may lead to high chlorophyll content when nitrogen is present in abundant supply; hence leaves of some –P plants display a dark green color [13], [16]. Being a mobile element, P can be reabsorbed from mature and senescing organs to young developing parts, hastening the senescence of older leaves [17]. Visual symptoms of P deficiency may be difficult to distinguish in some plants where some studies showed that Spathiphyllum grown in –P media for 110 days showed no visual symptoms although there was a reduction in chlorophyll content [4].

The increasing emphasis on non-destructive testing methods has prompted the development of devices like the chlorophyll meter which directly measures leaf transmittance and relates it with nitrogen status in the plant [18], [19]. A quantitative analysis of the in vivo fluorescence kinetics, called ‘JIP-test’, has been developed to analyze environmental effects on photosynthetic organisms [20]. Research has also focused on finding specific wavelengths or vegetation indices, from the leaf reflectance spectrum, which can characterize chlorophyll or pigment status [21], [22], [23], [24], [25]. In many studies, plants were subjected to prolonged periods of stress and spectral data were acquired at the point where physical stress symptoms became apparent. Such an approach precludes the use of such spectral indices for pre-visual determination of plant stress. In other work, seedlings were subjected to stress shortly after germination, a stage where the plants were most sensitive and would likely display pronounced visual symptoms very rapidly.

The aim of this study was to establish pre-visual spectral cues for Fe and P stress in plants at a more mature stage of growth. Instead of restricting observations to specific wavelengths, the whole visible spectrum (380–780 nm) was used to characterize the stress response. Since Fe is directly involved in electron transport processes and chlorophyll synthesis [26], [27], substantial spectral responses are expected and our aim was also to establish threshold values of the most sensitive spectral feature that can be used for pre-visual detection of Fe stress. In contrast, short term P-deficiency was not expected to result in drastic visual changes, but at best, subtle changes in chlorophyll content and thus would provide a challenging model to test the robustness of our spectral detection method. Physiological and morphological changes in relation to spectral changes were also tracked to evaluate the diagnostic value of optical signatures in assessing the nutritional status of plants.