Physiological and Morphological Responses of Blackberry Seedlings to Different Nitrogen Forms

Abstract

Blackberries are an emerging third-generation fruit that are popular in Europe, and specific nitrogen (N) supply is an important factor affecting their growth and development. To study the optimal N fertilizer for blackberry seedlings, no N (CK), nitrate (NO₃^-)-N, ammonium (NH₄⁺)-N and urea were applied to one-year-old 'Ningzhi 4' blackberry plants at a key growth period (from May to August) to explore the effects of different N forms on the physiological characteristics. Correlation and principal component analysis were used to determine the relationships between various indexes. Ammonium (NH₄⁺) or urea-fed plants had a better growth state, showed a greater plant height, biomass, SPAD values and enhanced antioxidant enzyme activities and photosynthesis. In addition, NH₄⁺ was beneficial to the accumulation of sugars and amino acids in leaves and roots, and promoted the transport of auxin and cytokinin to leaves. NO₃^- significantly inhibited root growth and increased the contents of active oxygen, malondialdehyde and antioxidants in roots. Correlation and principal component analysis showed that growth and dry matter accumulation were closely related to the antioxidant system, photosynthetic characteristics, amino acids and hormone content. Our study provides a new idea for N regulation mechanism of blackberry and proposes a scientific fertilization strategy.

Keywords: ammonium; blackberry; nitrate; nitrogen; physiological parameters; plant growth.

Figure 1

(A) Plant height and main stem diameter in response to different N forms. (B) The dry weights of the roots, shoots and whole plants in response to different N forms. (C) Phenotypes of the above- and below-ground parts of blackberry after 60 days of treatment with different N forms. Different letters show significant differences between treatments (uppercase letters indicate p < 0.01, lowercase letters indicate p < 0.05).

Figure 2

Scanning electron micrographs showing the leaf cross-sections ((A–D); scale bar is 200 μm), leaf upper surface ((E–H); scale bar is 50 μm), leaf lower surface ((I–L); scale bar is 50 μm) and root surface ((M–P); scale bar is 200 μm) after 60 days of treatment with different N forms. The counts of UEC (Q), GH (R), stomata (S) and OS (T) with different N forms. X-ray energy spectrogram of leaf cross-sections with different N treatments (panels (U–X) represent the CK, NH₄⁺–N, NO₃⁻–N and urea treatments, respectively). PTC, palisade tissue cell; STC, spongy tissue cell; UEC, upper epidermal cell; GH, glandular hairs; OS, open stomata.

Figure 3

Changes in root physiology and antioxidant indicators with different N forms. The MDA (A), H₂O₂ (C), AsA (G), GSH (H) and SP (I) levels. O₂^·− generation rate of (B). Activities of SOD (D), POD (E) and CAT (F). MDA, malondialdehyde; O₂^·−, superoxide anion radical; H₂O₂, hydrogen peroxide; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; AsA, ascorbic acid; GSH, reduced glutathione; SP, soluble protein. The data indicated are the means ± SDs (n = 3). Different letters show significant differences between treatments (** p < 0.01, * p < 0.05).

Figure 4

Effects of different N forms on the leaf photosynthetic parameters of blackberry. Changes in the relative chlorophyll content (SPAD value) and N content with time (A). Changes in air temperature (AT) and atmospheric CO₂ concentration (Ca) during the day (B). Effects of different N forms on the net photosynthetic rate (Pn) and transpiration rate (Tr) (C), intercellular CO₂ concentration (Ci) and stomatal conductance (Gs) (D), leaf water use efficiency (LWUE) and light use efficiency (LUE) (E) and stomatal limitation (Ls) (F). The data indicated are the means ± SDs (n = 20). Different letters show significant differences between treatments (uppercase letters indicate p < 0.01, lowercase letters indicate p < 0.05).

Figure 5

Effects of different N forms on sugar (A–D) and endogenous hormone contents (E,F) in roots and leaves. The data presented are the means ± SDs (n = 3). Different letters show significant differences between treatments (uppercase letters indicate p < 0.01, lowercase letters indicate p < 0.05).

Figure 6

(A) Correlation matrix of the physiological indicators. The results were derived from the Pearson correlation analysis. NC, nitrogen content; RSS, soluble sugar in roots; LSS, soluble sugar in leaves; RCTK, cytokinin in roots; LCTK, cytokinin in leaves; RIAA, auxin in roots; LIAA, auxin in leaves; RTAA, total amino acid in roots; LTAA, total amino acid in leaves. * represents a significant correlation at the 0.05 level and ** represents a significant correlation at the 0.01 level. (B) PCA score chart of the physicochemical properties of blackberry with different N forms.

Figure 7

Physiological characteristics of whole blackberry plants with different N forms affected blackberry growth and the associated correlations: a possible physiological regulatory mechanism. The red font indicates that an indicator has a significant positive correlation with blackberry growth and the green font indicates a negative correlation. The upward arrow indicates that the value of this indicator increases with the NH₄⁺–N treatment relative to the NO₃⁻–N treatment, that is, NH₄⁺/NO₃⁻ > 1; the downward arrow indicates the opposite. * represents p < 0.05, and ** represents p < 0.01.