Yong-Zhong Liu

Boron deficiency in woody plants: various responses and tolerance mechanisms

Boron (B) is an essential microelement for higher plants, and its deficiency is widespread around the world and constrains the productivity of both agriculture and forestry. In the last two decades, numerous studies on model or herbaceous plants have contributed greatly to our understanding of the complex network of B-deficiency responses and mechanisms for tolerance. In woody plants, however, fewer studies have been conducted and they have not well been recently synthesized or related to the findings on model species on B transporters. Trees have a larger body size, longer lifespan and more B reserves than do herbaceous plants, indicating that woody species might undergo long-term or mild B deficiency more commonly and that regulation of B reserves helps trees cope with B deficiency. In addition, the highly heterozygous genetic background of tree species suggests that they may have more complex mechanisms of response and tolerance to B deficiency than do model plants. Boron-deficient trees usually exhibit two key visible symptoms: depression of growing points (root tip, bud, flower, and young leaf) and deformity of organs (root, shoot, leaf, and fruit). These symptoms may be ascribed to B functioning in the cell wall and membrane, and particularly to damage to vascular tissues and the suppression of both B and water transport. Boron deficiency also affects metabolic processes such as decreased leaf photosynthesis, and increased lignin and phenol content in trees. These negative effects will influence the quality and quantity of wood, fruit and other agricultural products. Boron efficiency probably originates from a combined effect of three processes: B uptake, B translocation and retranslocation, and B utilization. Root morphology and mycorrhiza can affect the B uptake efficiency of trees. During B translocation from the root to shoot, differences in B concentration between root cell sap and xylem exudate, as well as water use efficiency, may play key roles in tolerance to B deficiency. In addition, B retranslocation efficiency primarily depends on the extent of xylem-to-phloem transfer and the variety and amount of cis-diol moieties in the phloem. The B requirement for cell wall construction also contribute to the B use efficiency in trees. The present review will provide an update on the physiological and molecular responses and tolerance mechanisms to B deficiency in woody plants. Emphasis is placed on the roles of B reserves that are more important for tolerance to B deficiency in trees than in herbaceous plants and the possible physiological and molecular mechanisms of differential B efficiency in trees. We propose that B may be used to study the relationship between the cell wall and the membrane via the B-bridge. Transgenic B-efficient tree cultivars have considerable potential for forestry or fruit rootstock production on low B soils in the future.

Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray

Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

Citrus PH5-like H(+)-ATPase genes: identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits.

PH5 is a petunia gene that encodes a plasma membrane H+-ATPase and determines the vacuolar pH. The citrate content of fruit cell vacuoles influences citrus organoleptic qualities. Although citrus could have PH5-like homologs that are involved in citrate accumulation, the details are still unknown. In this study, extensive data-mining with the PH5 sequence and PCR amplification confirmed that there are at least eight PH5-like genes (CsPH1-8) in the citrus genome. CsPHs have a molecular mass of approximately 100 kDa, and they have high similarity to PhPH5, AtAHA10 or AtAHA2 (from 64.6 to 80.9%). They contain 13–21 exons and 12–20 introns and were evenly distributed into four subgroups of the P3A-subfamily (CsPH1, CsPH2, and CsPH3 in Group I, CsPH4 and CsPH5 in Group II, CsPH6 in Group IV, and CsPH7 and CsPH8 in Group III together with PhPH5). A transcript analysis showed that CsPH1, 3, and 4 were predominantly expressed in mature leaves, whereas CsPH2 and 7 were predominantly expressed in roots, CsPH5 and 6 were predominantly expressed in flowers, and CsPH8 was predominantly expressed in fruit juice sacs (JS). Moreover, the CsPH transcript profiles differed between orange and pummelo, as well as between high-acid and low-acid cultivars. The low-acid orange “Honganliu” exhibits low transcript levels of CsPH3, CsPH4, CsPH5, and CsPH8, whereas the acid-free pummelo (AFP) has only a low transcript level of CsPH8. In addition, ABA injection increased the citrate content significantly, which was accompanied by the obvious induction of CsPH2, 6, 7, and 8 transcript levels. Taken together, we suggest that CsPH8 seems likely to regulate citrate accumulation in the citrus fruit vacuole.

Boron deficiency alters root growth and development and interacts with auxin metabolism by influencing the expression of auxin synthesis and transport genes

ABSTRACT Boron (B) deficiency inhibits and disturbs root growth and development by interacting with auxin (indole-3-acetic acid, IAA). However, the underlying mechanism of this interaction is still poorly understood. This study found that plants in a long-term boron deprivation treatment (∼0.25 µg L−1) had inhibited elongation of trifoliate orange roots, enlarged root tips and severe necrosis as well as a significant decrease in soluble boron and IAA content in seedling root tips. The results of a short-term boron deprivation treatment showed that a significant decrease in soluble boron and IAA content occurred after 3 hours of treatment (HOT) and 1 day of treatment (DOT), respectively. Moreover, the expression of IAA synthetic genes (TAA1, TAR2, YUC3 and YUC8) was strongly induced as early as 3 HOT and was then significantly reduced. The expression of rootward IAA transport genes (AUX1, PIN1 and PIN4) decreased significantly in the boron deprivation treatment, but the expression levels of shootward IAA transport genes (LAX1, ABCB1 and PIN3) were significantly increased. Taken together, the increase in IAA content before 1 DOT may be due to increased IAA synthesis caused by the induction of TAA1, TAR2, YUC3 and YUC8 expression; the subsequent decrease and the significantly lower final IAA content compared to the +B treatment (0.25 mg L−1 B) may be due to the reduced expression of IAA synthetic genes and rootward IAA transport genes and the increased expression of shootward IAA transport genes. Abbreviations: DAT: days after treatment; DOT: day of treatment; FW: fresh weight; HOT: hour of treatment; isB: insoluble boron; LC: liquid chromatography; LSD: least-significant difference; MS: mass spectrometry; PAT: polar auxin transport; qRT-PCR: quantitative real-time polymerase chain reaction; sB: soluble boron

Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’.

Physiological and transcriptional analysis reveals pathways involved in iron deficiency chlorosis in fragrant citrus

Iron (Fe) deficiency chlorosis is a yield-limiting problem in citrus production regions with calcareous soils. Physiological and transcriptional analyses of fragrant citrus (Citrus junos Sieb. ex Tanaka) leaves from Fe-sufficient (IS) and Fe-deficient (ID) plants were investigated in this study. The physiological results showed that Fe, potassium, and nitrogen levels decreased by 12, 15, and 41% in ID leaves, respectively. However, zinc and copper levels increased by 49 and 35% in ID leaves, respectively. The chlorophyll (Chl) content, photosynthesis rate, stomatal conductance, and transpiration rate in ID leaves decreased by 55, 33, 38, and 42%, respectively, compared with IS leaves. Moreover, transcriptional profiling analysis showed that genes associated with Chl metabolism, photosynthesis, and nitrogen metabolism were dramatically downregulated by Fe deficiency. The expression of glutamyl-tRNA reductase 1, chlorophyll(ide) b reductase, and geranylgeranyl diphosphate reductase in ID leaves was 0.26–0.37 times that in IS leaves. The expression levels of 16 photosynthesis-related genes were severely downregulated by Fe deficiency. In addition, the transcription levels of nitrate transporter, nitrate reductase, and ferredoxin-nitrite reductase genes in ID leaves were 0.38–0.45 times those in IS leaves. Taken together, these results indicated that the block of Chl biosynthesis, the reduction of photosynthesis, and the repression of nitrogen absorption resulted in the chlorosis symptoms observed in fragrant citrus leaves.

Transcriptome Changes Associated with Boron Deficiency in Leaves of Two Citrus Scion-Rootstock Combinations

Boron (B) deficiency stress is frequently observed in citrus orchards and causes considerable loss of productivity and fruit quality. Carrizo citrange (Cc) has been reported as a rootstock more tolerant to B deficiency than Trifoliate orange (To). The ‘Newhall’ navel orange (Ns) performed better when grafted onto Cc (Ns/Cc) than when grafted onto To (Ns/To) under long-term B deficiency. The present study confirmed that Ns/Cc had higher boron content, leaf fresh weight, lower leaf chlorosis and stronger photosynthesis ability than Ns/To. Moreover, B-deficiency significantly reduced the chlorophyll and carotenoid content in Ns/To. The content of total soluble sugar and lignin were dramatically increased and the expression levels of photosynthesis-related genes were substantially down-regulated in Ns/To by B-deficient treatment. B-deficiency also strongly induced expression levels of chlorophyll decomposition-related genes, glucose synthesis-related genes and lignin synthesis-related genes, and significantly inhibited the expression of carotenoid synthesis-related genes in Ns/To. Overall, these findings suggested that the influence of To on the scion of Ns was worse than that of Cc due to differently regulating these metabolic pathways under the long term of B-deficiency. The transcriptome analysis provided further information for understanding the mechanism of the different responses of scion-rootstock combinations to B-deficiency stress.