Influence of thylakoid membrane lipids on the structure and function of the plant photosystem II core complex

Main conclusion

MGDG leads to a dimerization of isolated, monomeric PSII core complexes. SQDG and PG induce a detachment of CP43 from the PSII core, thereby disturbing the intrinsic PSII electron transport. The influence of the four thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure and function of isolated monomeric photosystem (PS) II core complexes was investigated. Incubation with the negatively charged lipids SQDG and PG led to a loss of the long-wavelength 77 K fluorescence emission at 693 nm that is associated with the inner antenna proteins. The neutral galactolipids DGDG and MGDG had no or only minor effects on the fluorescence emission spectra of the PSII core complexes, respectively. Pigment analysis, absorption and 77 K fluorescence excitation spectroscopy showed that incubation with SQDG and PG led to an exposure of chlorophyll molecules to the surrounding medium followed by conversion to pheophytin under acidic conditions. Size-exclusion chromatography and polypeptide analysis corroborated the findings of the spectroscopic measurements and pigment analysis. They showed that the negatively charged lipid SQDG led to a dissociation of the inner antenna protein CP43 and the 27- and 25-kDa apoproteins of the light-harvesting complex II, that were also associated with a part of the PSII core complexes used in the present study. Incubation of PSII core complexes with MGDG, on the other hand, induced an almost complete dimerization of the monomeric PSII. Measurements of the fast PSII fluorescence induction demonstrated that MGDG and DGDG only had a minor influence on the reduction kinetics of plastoquinone Q_A and the artificial PSII electron acceptor 2,5-dimethyl-p-benzoquinone (DMBQ). SQDG and, to a lesser extent, PG perturbed the intrinsic PSII electron transport significantly.     

Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids

The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after ¹⁴CO₂ feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as “carriers” for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after ¹⁴CO₂ and ¹⁴C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids.     

Aluminum mediates compositional alterations of polar lipid classes in maize seedlings

Changes in lipid composition were investigated on maize roots and shoots under aluminum stress. After 4d exposure to 100 microM Al, root growth was inhibited while shoot growth was not affected. In roots, the decrease of the DBI (double bond index) of total fatty acids may signal a decrease in membrane fluidity. The total lipids (TL) decreased by 49%, but phospholipids (PL), phosphatidylcholine (PC) and phosphatidylinositol (PI) increased to approximately 3-fold. The MGDG increased to 2-fold but no significant change was found in the DGDG. The steryl lipids (SL) increased by 69%. The SL/PL ratio decreased from 2.64 to 1.52 and the MGDG/DGDG ratio increased from 0.45 to 1.06 in roots of Al-stressed plants. Al leads to oxidative stress in roots of treated plants as indicated by the increase of malondialdehyde (MDA) concentrations. In shoots, changes in fatty acid composition were associated with an increase of the DBI in all lipid classes except that of the DGDG decreased. The PG was the lipid class which shows the large variation of fatty acid composition. No significant changes were found either for TL, PL, SL or MDA concentrations in shoots of Al-treated plants. While PE levels did not show significant change, PI and PG increased and PC decreased. However, the Al caused 87% decrease in the GL levels. The MGDG and DGDG decreased to 19- and 8-fold, respectively. The deleterious effects of Al on polar lipids could be caused by a direct intervention of Al on plasma membrane and/or alteration of cell metabolism.     

Galactolipid Synthesis in Vicia faba Leaves: I. Galactose, Glycerol, and Fatty Acid Labeling after CO(2) Feeding

The galactose, glycerol, and fatty acids of mono- and digalactosyl diglycerides (MGDG and DGDG) have been separated and analyzed for ¹⁴C activity after ¹⁴CO₂ feeding of Vicia faba leaf discs. Fully expanded and developing leaves were analyzed at time intervals following feeding during continuous illumination. In addition, fully expanded leaves were analyzed after similar times in complete darkness. In all cases, ¹⁴C was incorporated very rapidly into galactose, whereas glycerol and fatty acids were labeled much more slowly and over a longer period of time. The data are consistent with the galactosylation of a diglyceride to MGDG which is in turn galactosylated to DGDG. The data suggest that the formation of diglycerides suitable for galactosylation to MGDG is slow in comparison to the galactosylation process. It is also suggested that DGDG may be formed from more than one pool of MGDG. The complete analysis of the ¹⁴C incorporation into galactose appears to represent the only satisfactory method of comparing galactolipid synthesis by ¹⁴C incorporation. Estimates of comparative rates of synthesis of MGDG and DGDG have been made on this basis.     

The effect of light intensity on galactolipid synthesis in Vicia faba leaves

The effect of light intensity upon galactolipid synthesis in Vicia faba leaf tissue was studied at two CO₂ concentrations, 0.03 and 1%. The rates of galactolipid synthesis were estimated by determining the amount of radioactivity in each of the two galactoses of digalactosyl diacylglycerol (DGDG) and the single galactose of monogalactosyl diacylglycerol (MGDG), a technique based upon the accepted pathway for galactolipid synthesis in which galactosylation is the terminal step in biosynthesis. The results suggest that the rates of MGDG and DGDG synthesis were similar under all conditions and that galactolipid synthesis was not directly affected by light intensity. The quantity of radioactivity incorporated into the galactoses of individual molecular species of MGDG and DGDG were similar under the light conditions used.     

Influence of thylakoid membrane lipids on the structure of aggregated light‐harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light‐harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana–stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X‐100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid‐induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana–stroma differentiation.     

Mono‐ and digalactosyldiacylglycerol composition of glaucocystophytes (Glaucophyta): A modern interpretation using positive‐ion electrospray ionization/mass spectrometry/mass spectrometry

Glaucocystophytes are freshwater algae that possess an almost‐intact cyanobacterium, referred to as a cyanelle, as their photosynthetic organelle. Because the cyanelle represents an intermediate state in plastid evolution, glaucocystophytes have been the subject of several studies to characterize the genetics and biochemistry of their cyanelles. However, only a small handful of older studies exist on the composition of their lipids, particularly two major plastid lipids, mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively), found in all photosynthetic life. Our study has used a modern mass spectrometry approach, namely positive‐ion electrospray ionization/mass spectrometry/mass spectrometry, to provide a fresh interpretation of the MGDG and DGDG composition of the species, Cyanophora paradoxa Korshikov and Glaucocystis nostochinearum Itzigsohn, representing two glaucocystophyte genera. We have found that the major forms of MGDG and DGDG (with sn‐1/sn‐2 regiochemistry) are 20:5/16:0 MGDG, 20:5/20:5 MGDG, 20:5/16:0 DGDG, and 20:5/20:5 DGDG. A comparison of these four forms, along with other more minor forms of MGDG and DGDG, to two examples of cyanobacteria has revealed that glaucocystophytes do not share intact forms of MGDG and DGDG with extant cyanobacteria, but may have maintained certain C₁₆ and C₁₈ cyanobacterial fatty acids.     

Gas-liquid chromatography of plant galactolipids and their deacylation and methanolysis products.

The gas-liquid chromatography of monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) and their deacylation and methanolysis products is reported. MGDG and DGDG and their galactosyl monoglycerides were chromatographed as their trimethylsilyl derivatives. Galactosyl monoglycerides were produced by partial deacylation of the diglycerides with Grignard's reagent and pancreatic lipase. The products of complete deacylation, mono- and digalactosyl glycerols, were separated as O-methyl, O-acetyl, O-trimethylsilyl and O-trifluoroacetyl derivatives. Gas-liquid chromatography of derivatives of the methanolysis products of MGDG and DGDG and the methylated galactosyl glycerols allowed the separation and quantitative recovery of the galactose and glycerol of both lipids and the two galactoses of DGDG.     

Incorporation of14C-radioactivity into various lipid classes at different growth-stages of culturedLaminaria japonica

A study is reported on the incorporation of¹⁴C-acetate into lipid classes from three different growth stages ofLaminaria japonica, a species long used for food in Japan. This was done because of the possible utilization of its lipids.Radioactivity incorporated into whole lipids in the three growth stages under the same experimental conditions (10 °C, 500 lux) increased with maturity of the thalli. The radioactivity was found mainly in PC, TG and 1,2-DG and subsequently distributed into other lipid classes (PG,PI,PE,MGDG,SQDG and DGDG) to a lesser extent. The incorporation patterns of the former group were similar at all stages, but those of the latter group differed slightly according to growth stage.In juvenile thalli,¹⁴C was incorporated to a much higher extent into PG, MGDG, PI and fucosterol than PE, SQDG, DGDG and MG, while the¹⁴C-incorporation into MG, SQDG, DGDG and PS in the mature growth stage was higher than into the other lipid classes. The absolute level of incorporation was higher for all these compounds in mature thalli than the thalli of other growth stages.     

Plasma membrane lipid composition of the halophilic cyanobacterium Aphanothece halophytica

The plasma membrane from Aphanothece halophytica was isolated using both glycerol and sucrose gradient centrifugation. The isolated membrane was characterized for lipid content by TLC and isolated lipids were quantified by chemical analysis. The plasma membrane of A. halophytica was composed of MGDG, DGDG and PG. The sulfur containing lipid SQDG was not detected. The mole percent of each lipid in the plasma membrane varied with the external salinity of the media. MGDG was the most abundant lipid in the plasma membrane of cells grown at one molar external NaCl. At three molar external NaCl, PG was the most abundant lipid. The ratio of uncharged to charged lipids comprising the plasma membrane decreased as the external salinity increased. It is possible that the alteration in lipid composition is of major importance in the adaptation of A. halophytica to changing external salinity.Abbreviations TLC Thin-layer chromatography - MGDG momogalactosyldiacylglycerol - DGDG digaloctosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulphoquinovosyldiacylglycerol     

Effects of Cadmium on Polar Lipid Composition and Unsaturation in Maize （Zea mays） in Hydroponic Culture

This study aimed to evaluate the effect of Cd exposure （100 μmol/L） on polar lipid composition, and to examine the level of fatty acid unsaturation in maize （Zea mays L.）. In roots, the level of 16：0 and monounsaturated fatty acids （16：1 ＋ 18：1） decreased in phosphatidylcholine （PC） and phosphatidylethanolamine （PE）. In contrast, the proportion of unsaturated 18-C fatty acid species showed an opposite response to Cd. The content, on the other hand, of PC, PE, digalactosyldiacylglycerol （DGDG）, and steryl lipids increased in roots （2.9-, 1.6-, 5.3-, and 1.7-fold increase, respectively）. These results suggest that a more unsaturated fatty acid composition than found in control plants with a concomitant increase in polar lipids may favor seedling growth during Cd exposure. However, the observed increase in the steryl lipid （SL） ： phospholipid （PL） ratio （twofold）, the decrease in monogalactosyldiacylglycerol （MGDG） ： DGDG ratio, as well as the induction of lipid peroxidation in roots may represent symptoms of membrane injury. In shoots, the unsaturation level was markedly decreased in PC and phosphatidylglycerol （PG） after Cd exposure, but showed a significant increase in sulfoquinovosyldiacylglycerol （SQDG）, MGDG and DGDG. The content of PG and MGDG was decreased by about 65%, while PC accumulated to higher levels （4.4-fold increase）. Taken together, these changes in the polar lipid unsaturation and composition are likely to be due to alterations in the glycerolipid pathway. These results also support the idea that the increase in overall unsaturation plays some role in enabling the plant to withstand the metal exposure.     

The Effect of Light Intensity, Day Length, and Temperature on Fatty Acid Synthesis and Desaturation in Vicia faba L.

Some effects of light intensity, day length, and temperatureon the fatty acid composition of the major glycerolipids ofleaves of Vicia faba L. (cv. Giant Windsor) were observed. Increasinglight intensity caused an increase in the relative concentrationsof 16 : 1 in PG and 18 : 3 in MGDG and DGDG. Increasing daylength during growth (and continuous illumination of leaf tissue)had no effect on 16 : 1 in PG but caused a decrease in the 18: 3 content of PG, PC, MGDG, and DGDG. Since the quantitiesof these lipids increased under these conditions, the decreasewas not due to photodestruction but to the differences in therelative rates of biosynthesis and desaturation of fatty acids.Incubation of leaf tissue in the dark for 4 d had little effecton the fatty acid composition of MGDG, DGDG, and PG. Temperaturealso controls fatty acid synthesis and desaturation. Above theoptimum growth temperature (20 °C), the 18 : 3 content ofMGDG, DGDG, PG, and PC decreased. In mature leaf tissue, thedegree of unsaturation of MGDG may be modified upward in responseto temperature changes. When plants were grown at 30 °Cand transferred to 20 °C the level of 18 : 3 in MGDG ofthe leaf tissue increased to levels found in plants grown onlyat 20 °C. The level of 18 : 3 in MGDG does not decreaseas rapidly when plants grown at 20 °C were transferred to30 °C. This suggests that the lower temperature induceddesaturation of 18 : 2 to 18 : 3.     

Effect of Temperature on Growth of Bacillus sp. T-4, a Thermophilic Acidophilic Bacterium

The lipid distribution and function in the thylakoid membranes from a thermophilic cyanobacterium, Mastigocladus laminosus, were investigated. The thylakoid membranes were treated with digitonin and separated on a DEAE-cellulose column into fractions enriched in photosystem I or II complex. Lipid analyses showed a specific distribution of anionic lipids among the fractions. A mild delipidation of the membranes with cholate indicates that monogalactosyl diacylglycerol (MGDG) and sulfoquinovosyl diacylglycerol (SQDG) are released rapidly, while the major parts of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG) are tightly associated with membranes, suggesting a different distribution between the two groups of lipids. Measurements of fluorescence of delipidated and reconstituted thylakoids showed the contribution of lipids to energy transfer. MGDG enhanced all the original fluorescence of thylakoids, while acidic PG and SQDG stimulated fluorescence of photosystem I and antena chlorophyll-protein complexes. DGDG was less effective under the conditions tested.     

Galactolipids and phospholipids of orange peel and juice chromoplasts

Chromoplasts from yellow orange (Citrus sinensis) fruit peel contain monogalactosyl diglycerides (MGDG), digalactosyl diglycerides (DGDG) and phosphatidyl glycerol (PG) in amounts similar to those found in chloroplasts from green fruit peel. Juice chromoplasts contain relatively little MGDG and no DGDG with high levels of phosphatidyl choline and phosphatidyl ethanolamine but no PG.     

Lipid Compositions of Photomixotrophic Green Calluses and Chlorophyll Deficient Leaves of Tobacco

Among photomixotrophic green calluses tested (N. rustica. N. tobacum L. cv. BY-4 and Samsun), the callus of Samsun had the highest contents of chlorophyll and chloroplast lipids, such as monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG), sulfoquinovosyldigly-ceride (SQDG) and phosphatidylglycerol (PG). However, the chlorophyll and chloroplast lipids in the green callus of Samsun were still 1/6 and 1/3 of that in the parent leaves, respectively. The relative content of a-linolenate in MGDG, DGDG and SQDG of the green calluses were higher than that of the white calluses. The ratios of hexadecatrienoate in MGDG and hexadeceno-ate (Δ³-trans) in PG in the green calluses were trace or less compared with that of the parent leaves. The crude lipids and total fatty acid contents of the chlorophyll deficient leaves (N. taba-cum L. cv. Consolation 402 and Dominant Aurea Su/su) were almost the same as those of the normal leaves (cv. BY-4 and Samsun), although the chlorophyll contents of the chlorophyll deficient leaves were 1/3 ~ 1/4 of that of the normal leaves. The ratios of chloroplast lipids in the total polar lipids in the chlorophyll deficient leaves were a little lower than that in the normal green leaves, but the former had a slightly higher ratio of phospholipids such as phosphatidylcholine and phosphatidylethanolamine than the latter. There were few differences in the fatty acid compositions of each individual lipid betweeen both types of leaves.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

Lipids of Ectocarpus fasciculatus (Phaeophyceae). Incorporation of [l-14C]Oleate and the Role of TAG and MGDG in Lipid Metabolism

Lipids and fatty acids of Ectocarpus fasciculatus (Ectocarpales,Phaeophyceae) were analyzed. Major polar lipids are monogalactosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol(SQDG), diacylglycerylhydroxymethyl-N,N,N-trimethyl-rß-alanine(DGTA), phosphatidylcholine (PC), phospha-tidylethanolamine(PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI).Diphosphatidylglycerol (DPG), phosphatidic acid (PA) and phosphatidyl-O-[N-(2-hydroxy-ethyl)glycine](PHEG) were also present in small amounts. Nonpolar lipids mainlyconsist of triacylglycerol (TAG) and diacylglycerol (DAG). Majorfatty acids are 16:0,18:1, 18:3, 18:4, 20:4 and 20:5. The positionaldistribution of fatty acids showed that molecular species ofeukaryotic structure account for 99% in MGDG, 98% in DGDG, 62%in PG and 23% in SQDG. On incubation with [1-¹⁴C]18:1 for 30min, 33% of the total label was detected in TAG, 16% in PG,14% in PE, 10% in PC and 8% in MGDG. During 7 days of chase,the label in TAG, PG, PE and PC decreased and simultaneouslyincreased in MGDG up to 41% of the total. In SQDG, labelledfatty acids were found in prokaryotic as well as in eukaryoticmolecular species. During the experiment, the label shiftedfrom 18:1 to 18:2, 18:3, 18:4 and, to a minor extent, to 20:4and 20:5 acids indicating 18:1 to be processed by elongationand/or desaturation. These results suggest TAG to act as a majorprimary acceptor of exogenous oleate and to be involved in thetransfer of fatty acids to MGDG and other polar lipids. (Received March 24, 1997; Accepted June 11, 1997)     

膜脂对菠菜细胞色素b6f复合体电子传递活性的影响

利用从菠菜(Spinacia oleracea L.)叶绿体分离、纯化出的缺失膜脂的细胞色素b6f蛋白复合体(Cyt b6f)制剂与从菠菜类囊体分离、纯化的膜脂进行体外重组,检测了不同膜脂对Cyt b6f催化电子传递活性的影响.结果表明:被检测的5种膜脂,即单半乳糖基甘油二酯(MGDG)、双半乳糖基甘油二酯(DGDG)、磷脂酰胆碱(PC)、磷脂酰甘油(PG)和硫代异鼠李糖基甘油二酯(SQDG)对Cyt b6f催化电子传递的活性均有明显的促进作用,但促进的程度各不相同,这可能与这些膜脂分子的带电性质密切相关.不带电荷的MGDG和DGDG及分子整体呈电中性的PC对促进Cyt b6f催化电子传递的活性非常有效,可分别使其活性提高89%、75%和77%;而带负电荷的PG和SQDG对活性的促进作用则相对较弱,仅可使其活性分别提高43%和26%.     

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.