Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition

Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of (15)N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early- and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.     

Natural variation in the regulation of leaf senescence and relation to N and root traits in wheat

Objectives

To identify parameters that can be used for the analysis of natural variation in leaf senescence of wheat; and to understand the association between the onset and progression of leaf senescence with N uptake and root traits.

Methods

Chlorophyll content and the proportion of yellow leaves were used as senescence indicators and their relation with other morphological and physiological traits were measured in contrasting early senescing (ES) and late senescing (LS) wheat lines.

Results

There were significant genotype effects on the onset and progress of senescence. The ES lines in which leaf senescence commenced early had significantly lower root biomass and N uptake than LS lines. The strong negative association between the extent of leaf senescence with root biomass and N uptake indicated that the poor root growth induced N limitation caused the early senescence of ES lines.

Conclusions

The leaf senescence development in ES lines was precocious and constitutive as the trait expressed even under optimal growth conditions suggesting they could be useful in understanding the genetic regulation of senescence under different abiotic stress situations. Accelerated leaf senescence in wheat could be a mechanism to compensate for limitations in the root system that tend to restrict nutrient uptake.     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean

Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.     

Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in arabidopsis.

Factors that influence the longevity and senescence of photosynthetic tissues of Arabidopsis were investigated. To determine the influence of reproductive development on the timing of somatic tissue senescence, the longevity of rosette leaves of the Landsberg erecta strain and of isogenic mutant lines in which flowering is delayed (co-2) or sterile flowers are produced (ms1-1) were compared. No difference in the timing of senescence of individual leaves was observed between these lines, indicating that somatic tissue longevity is not governed by reproductive development in this species. To examine the role of differential gene expression in the process of leaf senescence, cDNA clones representing genes that are differentially expressed in senescing tissues were isolated. Sequence analysis of one such clone indicated homology to previously cloned cysteine proteinases, which is consistent with a role for the product of this gene in nitrogen salvage. RNA gel blot analysis revealed that increased expression of senescence-associated genes is preceded by declines in photosynthesis and in the expression of photosynthesis-associated genes. A model is presented in which it is postulated that leaf senescence is triggered by age-related declines in photosynthetic processes.     

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence.     

毛竹叶片衰老过程的叶重量、叶面积及元素内吸收率的动态

对福建永春毛竹(Phyllostachys pubescens Mazel ex H.de Lehaie)叶片衰老过程的叶重量、叶面积及元素内吸收率的动态进行了研究,并对元素内吸收率RE_1(以元素的干重含量为计算单位,mg/g)、RE_2(以单位叶片的元素含量为计算单位,mg/leaf)以及RE_3(以单位叶面积的元素含量为计算单位,mg/cm~2)进行了比较。叶片衰老过程中,平均叶重量、叶面积及比叶重分别下降了19.55％、15.16％和5.07％。叶重量与叶面积下降百分率的季节变化趋势一致,说明毛竹叶片存在一定的重量与面积比率。在不同的元素内吸收率比较中,N和K的元素内吸收率均为正,Ca均为负,表明叶片衰老过程中N和K的元素含量从衰老叶片中转移至植株的其他部位,而Ca在老叶中累积。N、P、K、Ca和Mg 5种元素平均的元素内吸收率高低顺序均为RE_2>RE_3>RE_1,反映出以元素的干重含量为计算单位和以单位叶面积的元素含量为计算单位的元素内吸收率偏低。     

毛竹叶片衰老过程的叶重量、叶面积及元素内吸收率的动态

对福建永春毛竹(Phyllostachyspubescens Mazel ex H.de Lehaie)叶片衰老过程的叶重量、叶面积及元素内吸收率的动态进行了研究,并对元素内吸收率RE1(以元素的干重含量为计算单位,mg/g)、RE2(以单位叶片的元素含量为计算单位,mg/leaf)以及RE3(以单位叶面积的元素含量为计算单位,mg/cm2)进行了比较.叶片衰老过程中,平均叶重量、叶面积及比叶重分别下降了19.55%、15.16%和5.07%.叶重量与叶面积下降百分率的季节变化趋势一致,说明毛竹叶片存在一定的重量与面积比率.在不同的元素内吸收率比较中,N和K的元素内吸收率均为正,Ca均为负,表明叶片衰老过程中N和K的元素含量从衰老叶片中转移至植株的其他部位,而Ca在老叶中累积.N、P、K、Ca和Mg5种元素平均的元素内吸收率高低顺序均为RE2＞RE3＞RE1,反映出以元素的干重含量为计算单位和以单位叶面积的元素含量为计算单位的元素内吸收率偏低.     

Senescence-associated mRNAs that may participate in signal transduction and protein trafficking

The differential display technique was used to generate cDNA probes in order to identify mRNAs that are up-regulated during senescence of Arabidopsis leaves. Three mRNAs were examined that had not previously been associated with senescence. The steady-state levels of these mRNAs are detectable in small amounts in mature green leaves, but increase considerably as chlorophyll levels begin to decline. This relationship to senescence occurs under natural circumstances as well as when senescence is accelerated by leaf detachment in the dark or by addition of 1-aminocyclopropane-1-carboxylic acid (ACC). Retardation of senescence by benzyladenine slows the increase of the mRNAs. One of these mRNAs appears to code for a protein (Sec 13) that may be involved in vesicle formation at the endoplasmic reticulum. Another mRNA codes for a protein with WD‐repeat motif whose function is as yet unidentified, and the third codes for a putative calcium-dependent protein kinase. A fourth cDNA has also been cloned by subtractive hybridization from senescing Arabidopsis leaves that encodes vacuolar-processing enzyme ( γ VPE). Incubation of detached leaves in darkness also caused an abrupt elevation in the steady-state levels of the γVPE , similar to that of the senescing attached leaves. The possible functions of the gene products and their involvement in cellular and biochemical processes during senescence are discussed.     

Premature leaf senescence modulated by the Arabidopsis PHYTOALEXIN DEFICIENT4 gene is associated with defense against the phloem-feeding green peach aphid

Aphids, which are phloem-feeding insects, cause extensive loss of plant productivity and are vectors of plant viruses. Aphid feeding causes changes in resource allocation in the host, resulting in an increase in flow of nutrients to the insect-infested tissue. We hypothesized that leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf, could be utilized by plants to limit aphid growth. Using Arabidopsis (Arabidopsis thaliana) and green peach aphid (GPA; Myzus persicae Sulzer), we found that GPA feeding induced premature chlorosis and cell death, and increased the expression of SENESCENCE ASSOCIATED GENES (SAGs), all hallmarks of leaf senescence. Hypersenescence was accompanied by enhanced resistance against GPA in the Arabidopsis constitutive expresser of PR genes5 and suppressor of SA insensitivity2 mutant plants. In contrast, resistance against GPA was compromised in the phytoalexin deficient4 (pad4) mutant plant. The PAD4 gene, which is expressed at elevated level in response to GPA feeding, modulates the GPA feeding-induced leaf senescence. In comparison to the wild-type plant, GPA feeding-induced chlorophyll loss, cell death, and SAG expression were delayed in the pad4 mutant. Although PAD4 is associated with camalexin synthesis and salicylic acid (SA) signaling, camalexin and SA signaling are not important for restricting GPA growth; growth of GPA on the camalexin-biosynthesis mutant, pad3, and the SA deficient2 and NahG plants and the SA-signaling mutant, nonexpresser of PR genes1, were comparable to that on the wild-type plant. Our results suggest that PAD4 modulates the activation of senescence in the aphid-infested leaves, which contributes to basal resistance to GPA.     

Natural allelic variation in the temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock

Temperature compensation is a defining feature of circadian oscillators, yet no components contributing to the phenomenon have been identified in plants. We tested 27 accessions of Arabidopsis thaliana for circadian leaf movement at a range of constant temperatures. The accessions showed varying patterns of temperature compensation, but no clear associations to the geographic origin of the accessions could be made. Quantitative trait loci (QTL) were mapped for period and amplitude of leaf movement in the Columbia by Landsberg erecta (CoL) and Cape Verde Islands by Landsberg erecta (CvL) recombinant inbred lines (RILs) at 12 degrees , 22 degrees , and 27 degrees . Six CvL and three CoL QTL were located for circadian period. All of the period QTL were temperature specific, suggesting that they may be involved in temperature compensation. The flowering-time gene GIGANTEA and F-box protein ZEITLUPE were identified as strong candidates for two of the QTL on the basis of mapping in near isogenic lines (NILs) and sequence comparison. The identity of these and other candidates suggests that temperature compensation is not wholly determined by the intrinsic properties of the central clock proteins in Arabidopsis, but rather by other genes that act in trans to alter the regulation of these core proteins.